Tissue-derived extracellular vesicles and their use as diagnostics

ABSTRACT

The present disclosure relates to a method of isolating extracellular vesicles directly from human tissues. The invention further relates to a method of identifying disease and tissue specific membrane proteins on extracellular vesicles by membrane isolation and proteomic analysis. The invention further relates to methods of diagnosing diseases by capturing extracellular vesicles by the use of disease specific membrane proteins from body fluids, and detecting or analyzing molecular signatures (proteome, DNA, and RNA) on captured extracellular vesicles. Moreover, the present invention relates to kits, apparatus and software required for implementing aforementioned methods.

TECHNICAL FIELD

The present disclosure relates to apparatus and methods forextracellular vesicles-based diagnosis of diseases, in particularapparatus and methods for identifying membrane proteins on extracellularvesicles which are specific for diseases and diagnosing diseases byusing them. Moreover, the present disclosure relates to methods ofisolating extracellular vesicles from tissues and identifying membraneproteins by membrane isolation, so called membrane proteomics.Furthermore, the present disclosure also relates to computer programproducts comprising non-transitory (namely non-transient)computer-readable storage media having computer-readable instructionsstored thereon, the computer-readable instructions being executable by acomputerized device comprising processing hardware to execute theaforesaid methods.

BACKGROUND

Extracellular vesicles are nano-sized vesicles (40 nm to 1000 nm indiameter) with a lipid bi-layer membrane and are known to be released bymany cells in the human body; extracellular vesicles include, forexample, exosomes, ectosomes, microvesicles and apoptotic bodies. Suchextracellular vesicles are operable to mediate intercellularcommunication by either activating surface receptors of recipient cellsor by transferring cargo proteins, nucleic acids (DNAs, mRNAs, siRNAs,miRNAs, and small non-coding RNAs), or lipids to recipient cells. One ofthe most attractive considerations for extracellular vesicles is that acargo of these extracellular vesicles is protected from extracellularenzymes and an immune system by a lipid membrane bilayer.

Extracellular vesicles have been found in many body fluids includingblood (plasma and serum), urine, ejaculate, saliva, cerebrospinal fluid,ascites, synovial fluid, bronchoalveolar lavage, pleural effusion,amniotic fluid, sweats, feces, cystic fluids, tears and breast milk, andthey are considered to carry signatures of the cells that produce them.This means that extracellular vesicles have a significant potential asfunctioning as diagnostic markers when seeking to detect diseases.Several extracellular vesicular markers have been proposed for use incancer diagnosis, including glypican-1 protein, EpCAM protein,KRAS-mutated DNA, and oncogenic mRNA and microRNAs.

Several cell types including reticulocytes, dendritic cells, B cells, Tcells, mast cells, epithelial cells, and embryonic cells are known to becapable of releasing exosomes, however their increased amount in theperipheral circulation appears to be unique to pregnancy and cancer.Furthermore, the primary source of circulating exosomes may beassociated with tumors. Tumor patients have been found to have very highlevels of tumor derived exosomes in plasma, ascites and pleuraleffusions.

To date, extracellular vesicular biomarker candidates have initiallybeen identified in extracellular vesicles from a cell line, and have insome cases been proposed to be valid also for clinical diagnosis, butare often not detectable in real clinical diseases. Thus, suchapproaches are not considered to be valid ways to identify extracellularvesicular biomarkers for human disease. Therefore, any molecularsignature of extracellular vesicles from human diseased tissue stillremains to be described.

SUMMARY

It is an object of the present disclosure to provide a new methodologyfor isolating extracellular vesicles from human tissues and identifyingmembrane and other extracellular vesicular molecular signatures asbiomarker candidates.

A further objective of the present disclosure is to use the membraneproteins, and other molecular signatures, in diagnosis of diseases.

These and other objects, which are evident to the skilled person fromthe present disclosure, are met by the different aspects of theinvention as claimed in the appended claims and as generally disclosedherein.

According to first aspect, the invention is realized by a method forisolating extracellular vesicles and identifying membrane proteinscomprising:

-   -   (a) providing human tissues;    -   (b) chopping the human tissues to make a plurality of pieces of        the human tissue;    -   (c) isolating the extracellular vesicles from the chopped pieces        of human tissue;    -   (d) treating the extracellular vesicles with an aqueous        solution, wherein the aqueous solution has a pH in a range of 9        to 14 to obtain membranes;    -   (e) isolating the membranes;    -   (f) identifying proteins on the membranes by employing mass        spectrometry to identify tissue and/or disease-specific membrane        proteins; and    -   (g) isolating extracellular vesicles with such specific membrane        proteins to identify at least one of:        -   i. their disease-specific proteome;        -   ii. their disease-related RNA cargo; and        -   iii. their disease-related DNA cargo.

Optionally, the method further comprises a step:

-   -   (h) incubating with enzymes to release the extracellular        vesicles from a tissue matrix, wherein the step h is performed        concomitantly after the step (a), (b), or (c).

The above defined method provides methods to isolate the extracellularvesicles from human tissues and to identify the membrane proteinsignatures for further analysis and biomarker discovery. All cells invivo produce the extracellular vesicles, and those extracellularvesicles are to a great extent entrapped in extracellular matrix oftissues. The released vesicles from any cell, or from any tissue,include a cloud of vesicles with different content, membrane moleculesand with cellular origin. This makes the extracellular vesiclesattractive biomarker sources. Current extracellular vesicles-basedmarkers are identified from cultured cells, not from actual in vivoorgan environment. Through the present invention, extracellular vesiclesare isolated directly from human tissues, which are more relevant andrealistic than cell culture-derived extracellular vesicles.

Optionally, the human tissues of steps (a) and (b) are obtained from ahuman body including tumors or diseased tissues. Further, the humantissues are derived from at least one of: liver, brain, kidney, heart,lung, skin, stomach, intestines, lymph nodes, bone marrow, adiposetissue, central and peripheral neural tissues, endocrine organs andmuscles.

Optionally, isolating the membranes at the step (e) includesultracentrifugation.

Optionally, the enzymes of the step (h) are one enzyme or combination ofenzymes that are chosen from proteases including matrixmetalloproteinase, collagenases, and papain and nucleases includingDNase, RNase, and Benzonase.

Optionally, the RNA and DNA cargo of the step g are mutated RNAs orDNAs, or non-mutated profiles of RNA and/or DNA sequences/genes.

According to an embodiment, the invention is realized by a method ofdiagnosing diseases using tissue or disease-specific membrane proteinson extracellular vesicles from body fluids, characterized in that themethod comprises steps of:

-   -   (a) isolating extracellular vesicles and identifying membrane        proteins using a method of first aspect;    -   (b) capturing extracellular vesicles by using an epitope        specific binder against the tissue or disease-specific membrane        proteins; and    -   (c) detecting extracellular vesicles by using an epitope        specific binder against the tissue or disease-specific membrane        proteins, or a combination of proteins.

Alternatively, the invention is realized by a method of diagnosingdiseases using tissue or disease-specific membrane proteins onextracellular vesicles obtained from body fluids, characterized in thatthe method comprises steps of:

-   -   (a) isolating extracellular vesicles and identifying membrane        proteins using a method of first aspect;    -   (b) capturing extracellular vesicles by using an        epitope-specific binder against the tissue or disease-specific        membrane proteins; and    -   (c) detecting nucleic acids on or in the captured extracellular        vesicles.

Alternatively, the invention is realized by a method of diagnosingdiseases using tissue and disease-specific membrane proteins onextracellular vesicles from body fluids, characterized in that themethod comprises steps of:

-   -   (a) isolating extracellular vesicles and identifying membrane        proteins using a method of first aspect;    -   (b) adding an epitope-specific binder against a tissue or        disease-specific membrane protein and a lipid-biotin conjugate        to sample; and    -   (c) detecting a tissue-specific membrane protein by using alpha        LISA donor and acceptor beads.

Optionally, the body fluids are blood (plasma and serum), urine,ejaculate, saliva, cerebrospinal fluid, ascites, synovial fluid, sputum,pleural effusion, amniotic fluid, sweats, feces, cystic fluids, tearsand breast milk, or lavage fluids from different body cavities such aslung, pleural cavity, stomach, small intestine, colon, nose, urinarybladder or the peritoneal cavity. More optionally, the epitope specificbinder is an antibody, an aptamer, or a peptide.

Optionally, the tissue or disease-specific membrane protein is a plasmamembrane protein. More optionally, the plasma membrane protein is HLA-DRprotein.

Optionally, the tissue or disease-specific membrane protein is amitochondrial membrane protein. More optionally, the mitochondrialmembrane protein is MTCO2 and/or COX6C proteins.

Optionally, the tissue or disease specific membrane protein is anendoplasmic reticulum membrane protein that includes at least one ofErlin-2 or RPN1 proteins.

Optionally, the diseases are cancers and/or inflammatory diseases. Moreoptionally, the nucleic acids are DNAs, mRNAs, miRNAs, ribosomal RNAs,and small non-coding RNAs and any other full length or fragment of RNAsor DNAs, including mutation-specific molecules.

Alternatively, the lipid-biotin conjugate is a cholesterol-polyethyleneglycol (PEG)-biotin.

According to a second aspect, the invention is realized by a kit forcapturing extracellular vesicles and detecting disease-associatedmarkers, characterized in that the kit comprises:

-   -   (a) an epitope specific binder against tissue or        disease-specific membrane proteins: and    -   (b) at least one disease-associated marker detection agent.

Optionally, the tissue or disease-specific membrane protein is a plasmamembrane protein, preferably HLA-DR protein.

Optionally, the tissue or disease-specific membrane protein is amitochondrial membrane protein. More optionally, the mitochondrialmembrane protein is MTCO2 and/or COX6C proteins. More optionally, thetissue or disease specific membrane protein is an endoplasmic reticulummembrane protein that includes at least one of Erlin-2 or RPN1 proteins.Optionally, the kit is adapted for use in any of the methods ofisolating extracellular vesicles and identifying membrane proteinsand/or diagnosing diseases using tissue or disease-specific membraneproteins on extracellular vesicles from body fluids.

According to third aspect, the invention is realized by an apparatus forimplementing the methods of isolating extracellular vesicles andidentifying membrane proteins and/or diagnosing diseases using tissue ordisease-specific membrane proteins on extracellular vesicles from bodyfluids.

According to fourth aspect, the invention is realized by a computerprogram product comprising non-transitory (namely non-transient)computer-readable storage media having computer-readable instructionsstored thereon, the computer-readable instructions being executable by acomputerized device comprising processing hardware to execute any of themethod of isolating extracellular vesicles and identifying membraneproteins and/or diagnosing diseases using tissue or disease-specificmembrane proteins on extracellular vesicles from body fluids.

It will be appreciated that features of the invention are susceptible tobeing combined in various combinations without departing from the scopeof the invention as defined by the appended claims.

A better understanding of the present invention may be obtained throughthe following examples which are set forth to illustrate, but are not tobe construed as limiting the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the present disclosure will be more fully understood fromexamples described herein below and the accompanying drawings, which isgiven by way of illustration only, and thus are not limitative of thepresent invention, and wherein:

FIG. 1 is an image wherein there is observed a presence of extracellularvesicles in interstitial space of a melanoma metastatic tissue, asrevealed by electron microscopy;

FIG. 2 is a schematic illustration of three different protocols forisolating the extracellular vesicles from melanoma metastatic tissues;

FIGS. 3A to 3F illustrate an RNA profile and morphology of extracellularvesicles isolated by three different protocols;

FIG. 4 is a Venn diagram of membrane proteins that are identified fromfive different extracellular vesicles, two from melanoma metastatictissues and three from cell lines, by mass spectrometry;

FIG. 5 is an illustration of a relative abundance comparison betweenmembrane proteins from melanoma or non-melanoma-derived extracellularvesicles;

FIG. 6 is an illustration of detection of cancer specific membraneproteins on isolated extracellular vesicles by ELISA;

FIG. 7 is a schematic illustration of sandwich ELISA to detect thedouble membrane proteins on extracellular vesicles;

FIG. 8 is an illustration of detection of double cancer specificmembrane proteins on isolated extracellular vesicles by sandwich ELISA;

FIG. 9 is an illustration of detection of double cancer specificmembrane proteins in plasma of melanoma patients and healthy control;

FIG. 10 is an illustration of detection of double cancer specificmembrane proteins on plasma of ovarian benign control, ovarian cancerpatients, and healthy control; and

FIG. 11 is an illustration of detection of double cancer specificmembrane proteins on cystic fluids of ovarian benign control and ovariancancer patients.

LIST OF ABBREVIATIONS

Abbreviation Meaning PBS Phosphate-buffered saline EDTAEthylenediaminetetraacetic acid BSA Bovine serum albumin MTCO2Mitochondrially encoded cytochrome c oxidase II SLC25A22 Solute CarrierFamily 25 Member 22 COX6C Cytochrome c oxidase subunit 6C EpCAMEpithelial cell adhesion molecule HLA-DR Human Leukocyte Antigen -antigen D Related HRP Horseradish peroxidase ELISA Enzyme-linkedimmunosorbent assay FACS Fluorescence-activated cell sorting LC-MS/MSLiquid chromatography tandem-mass spectrometry LFQ Label freequantification M Molar, mol/liter mM Millimolar, millimol/liter nmNanometer ml Milliliter DNA Deoxyribonucleic acid RNA Ribonucleic acidmRNA Messenger RNA miRNA Micro RNA siRNA Small interfering RNA HMC1Human mast cell line-1 MSC Mesenchymal stem cell MeT Metastatic tissueRPMI Roswell Park Memorial Institute ROC Receiver operatingcharacteristic AUC Area under curve H hour

Definitions

As used herein, the following terms shall have the following meanings:

As used herein, the term “extracellular vesicle” means a vesiclereleased by a cell. Examples of “extracellular vesicles” includeexosomes, ectosomes, microvesicles, prostasomes, oncosomes, andapoptotic bodies.

The term “membrane” means biological membranes, i.e. the outer coveringsof cells and organelles that allow passage of certain compounds. Whenthe term “membrane” is used as a noun herein, it typically refers to anextracellular vesicle or organelle which encloses an intravesicular ororganellar content and which has been opened to provide a non-enclosingform of the extracellular vesicle or organelle, i.e. a membrane form.Such membrane may originate from the outer Cell membrane, theGolgi-apparatus, the Endoplasmic reticulum, the nucleus or mitochondria.

The term “cancer” refers to a group of different diseases, which arecharacterized by unregulated cell growth and infiltration to neighboringtissues due to the disruption of programmed cell death. A target to bediagnosed according to the present invention may be selected from acancers selected from the group consisting of, but not limited to,carcinoma originating from epithelial cells, such as lung cancer, larynxcancer, stomach cancer, large intestine/rectal cancer, liver cancer,gallbladder cancer, pancreatic cancer, breast cancer, uterine cervicalcancer, prostate cancer, kidney cancer, skin cancer, or non-epithelialmalignancies such as malignant melanoma, sarcoma originating fromconnective tissue cells, such as bone cancer, muscle cancer, adiposetissue sarcoma, mesothelioma, etc., blood cancer originating fromhematopoietic cells or lymphatic system, such as leukemia, lymphoma,multiple myeloma, etc., and neuroma, neuroblastoma, medulloblastoma orany other tumor originating in peripheral or central nervous system.

The term “inflammatory diseases,” as used herein, refers to a syndromeor symptom including edema, resulting from an abnormal accumulation ofbody fluid in tissues, congestion due to vascular dilation, increasedheat by pyrogen and vasodilatation, and pain induced by arachidonic acidmetabolites. Inflammation may be classified as acute, sub-acute, andchronic inflammation according to time, and as infectious, allergic,auto-immune, toxic, metabolic and traumatic inflammatory diseasesaccording to pathophysiological conditions. A target to be diagnosedaccording to the present invention may be selected from the groupconsisting of, but not limited to, respiratory inflammatory diseasessuch as rhinitis, sinusitis, otitis media, rhinopharyngitis, laryngitis,bronchitis, asthma, chronic obstructive pulmonary disease, emphysema,bronchiectasis, bronchiolitis, pneumonia, pulmonary fibrosis, etc.,inflammatory diseases of the digestive system such as stomatitis,esophagitis, gastritis, peptic ulcer, irritable bowel syndrome,ulcerative colitis, cholecystitis, cholangitis, pancreatitis, hepatitis,etc., skin inflammation such as atopic dermatitis, psoriasis, etc.,cardiovascular inflammatory diseases such as endocarditis, myocarditis,pericarditis, vasculitis, arteriosclerosis, sepsis, etc., inflammatorydiseases of the endocrine system, such as thyroiditis, parathyroiditis,diabetes, etc., inflammatory diseases of the urogenital system such asnephritis, nephropathy, interstitial nephritis, orchitis, oophoritis,endometritis, vaginosis, etc., inflammatory diseases of themusculoskeletal system, such as rheumatoid arthritis, spondylarthritis,ostarthritis, gout, systemic lupus erythematosusethematosus, systemicsclerosis, myopathy, Sjogren syndrome, Behcet's disease,antiphospholipid syndrome, etc., inflammatory diseases of theNeuropsychiatric system, such as vascular dementia, Alzheimer's disease,degenerative brain diseases, depression, schizophrenia, and etc.

The terms “cell media”, “culture media” and/or “cell culture media” asused herein refer to a culture media used for preserving or culturingMelanoma metastatic tissues, cells and/or cell lines obtained frompatient during surgery. The culture media include all supplementsrequired for culturing and preservation of melanoma and/or carcinomacell lines. The culture media may not include fetal bovine serum. Forexample, if the sample is a solid sample, cells from the sample can becultured and exosome product is induced. In some embodiments, the sampleis ascites fluid from a subject, e.g., ascites fluid from a humansubject with ovarian cancer; cell culture media supernatant from a humanprimary melanoma cell line; cell culture mediasupernatant from a humanprimary colon cancer cell line; or murine macrophage, e.g., murinemacrophage infected with tuberculosis. The culture media may be anordinary medium, or may also be liquid nitrogen based medium. The liquidmedium can be isotonic, hypotonic, or hypertonic. In certainembodiments, the liquid medium contains a buffer and/or at least onesalt or a combination of salts. Buffers can maintain pH within aparticular range, for example, between 1 and 12, and are also referredto as pH stabilizing agents.

The term “membrane carrier” as used herein refers to membrane proteinsthat use electrochemical gradients to move selective chemical substratesacross lipid bilayers. The membrane carriers may be deep membranecarriers. Furthermore, the membrane carriers may include but not limitedto ions and small soluble organic molecules and/or lipid solublesubstrates. The membrane proteins that may candidates to be biomarkersare listed in Table-1.

The term “apoptoticbody” as used herein refers to degenerate basalepidermal cells. The apoptotic bodies may include but not limited tocolloid, hyaline, filamentous and/or civattebodies. Furthermore,theapoptotic bodies may be round, shrunken, homogeneous, eosinophilicbodies in the stratum basale.

DETAILED DESCRIPTION

The practice of the embodiments described in further detail below willemploy, unless otherwise indicated, conventional methods of diagnostics,molecular biology, cell biology, biochemistry and immunology within theskill of the art. Such techniques are explained fully in the literature.

It is appreciated that certain features of the invention, which are forclarity described in the context of separate embodiments may also beprovided in combination in a single embodiment. Conversely variousfeatures of the invention, which are for brevity, described in thecontext of a single embodiment, may also be provided separately and/orin any suitable sub-combination.

The methodology for isolating extracellular vesicles from human tissuesand identifying membrane and other extracellular vesicular molecularsignatures as biomarker candidates of the present disclosure is a methodof isolating extracellular vesicles and identifying membrane proteins,the method including steps of providing human tissues; chopping thehuman tissues to make a plurality of pieces of the human tissue;isolating the extracellular vesicles from the chopped pieces of humantissue; treating the extracellular vesicles with an aqueous solution,wherein the aqueous solution has a pH in a range of 9 to 14 to obtainmembranes; isolating the membranes;

identifying proteins on the membranes by employing mass spectrometry toidentify tissue and/or disease-specific membrane proteins; and isolatingextracellular vesicles with such specific membrane proteins to identifyat least one of their disease-specific proteome, their disease-relatedRNA cargo and their disease-related DNA cargo.

In an embodiment, the method of isolating extracellular vesicles andidentifying membrane proteins may include incubating the specificmembrane proteins with enzymes to release the extracellular vesiclesfrom a matrix just after providing the human tissues.

In another embodiment, the method of isolating extracellular vesiclesand identifying membrane proteins may include incubating the specificmembrane proteins with enzymes to release the extracellular vesiclesfrom a matrix just after chopping the human tissues.

In yet another embodiment, the method of isolating extracellularvesicles and identifying membrane proteins may include incubating thespecific membrane proteins with enzymes to release the extracellularvesicles from a matrix just after isolating the extracellular vesiclesfrom the chopped pieces of human tissue.

In an embodiment, the human tissues may include tumor or diseasedtissues and there may be no particular limitation on selection of humantissues. In an exemplary embodiment, the human tissues may be derivedfrom but not limited to liver, brain, kidney, heart, lung, skin,stomach, intestines, lymph nodes, bone marrow, adipose tissue andmuscles.

In an embodiment, isolating the membranes from the extracellularvesicles may include ultracentrifugation. For example, the membrane maybe isolated using density gradient ultracentrifugation.

In an embodiment, the enzymes used in incubation for releasing theextracellular vesicles from the matrix may include enzyme or combinationof enzymes chosen from proteases including matrix metalloproteinase,collagenases, and papain and nucleases including DNase, RNase, andBenzonase.

In another embodiment, identified disease-related RNA cargo or DNA cargoof the specific membrane proteins of the isolated extracellular vesiclesmay be mutated RNAs or DNAs.

The method of diagnosing diseases of the present disclosure is a methodof diagnosing diseases using tissue or disease-specific membraneproteins on extracellular vesicles from body fluids. The method includessteps of isolating extracellular vesicles and identifying membraneproteins, capturing extracellular vesicles by using an epitope specificbinder against the tissue or disease-specific membrane proteins anddetecting extracellular vesicles by using an epitope specific binderagainst the tissue or disease-specific membrane proteins, or acombination of proteins.

The method further includes steps of adding an epitope-specific binderagainst a tissue or disease-specific membrane protein and a lipid-biotinconjugate to sample and detecting a tissue-specific membrane protein byusing alpha capturing extracellular vesicles by using an epitopespecific binder against the tissue or disease-specific membrane proteinsdonor and acceptor beads.

In an embodiment, the method may include isolating the extracellularvesicles from body fluids for providing the human tissues.

In an embodiment, the body fluids may include but not limited to blood(plasma and serum), urine, ejaculate, saliva, cerebrospinal fluid,ascites, synovial fluid, sputum, pleural effusion, amniotic fluid,sweats, feces, cystic fluids, tears and breast milk, or lavage fluidsfrom different body cavities such as lung, pleural cavity, stomach,small intestine, colon, nose, urinary bladder or the peritoneal cavity.

In an embodiment, the epitope specific binder may be an antibody, anaptamer, and/or a peptide.

In another embodiment, the tissue or disease-specific membrane proteinmay be a plasma membrane protein. For example, the plasma membraneprotein may be HLA-DR protein and the tissue or disease-specificmembrane protein may be a mitochondrial membrane protein. In anexemplary embodiment, the mitochondrial membrane protein may be MTCO2and/or COX6C proteins.

In another embodiment, the tissue or disease specific membrane proteinmay be an endoplasmic reticulum membrane protein. In an exemplaryembodiment, the endoplasmic reticulum membrane proteins may be Erlin-2and/or RPN1 proteins.

In an embodiment, the diseases may be cancers and/or inflammatorydiseases, as described above.

In another embodiment, the nucleic acids may include but not limited toDNAs, mRNAs, miRNAs, ribosomal RNAs, and small non-coding RNAs and anyother full length or fragment of RNAs or DNAs, includingmutation-specific molecules.

In an exemplary embodiment, the lipid-biotin conjugate may be acholesterol-polyethylene glycol (PEG)-biotin.

The kit of the present disclosure for capturing extracellular vesiclesis a kit for capturing extracellular vesicles and detectingdisease-associated markers. The kit includes an epitope specific binderfor tissue or disease-specific membrane proteins and at least onedisease-associated marker detection agent.

In an embodiment, the tissue or disease-specific membrane protein may bea plasma membrane protein. For example, the plasma membrane protein maybe a HLA-DR protein.

In another embodiment, the tissue or disease-specific membrane proteinmay be a mitochondrial membrane protein. For example, the mitochondrialmembrane protein may be MTCO2 and/or COX6C proteins.

In yet another embodiment, the tissue or disease specific membraneprotein may be an endoplasmic reticulum membrane protein. For example,the endoplasmic reticulum membrane protein may be Erlin-2 and/or RPN1proteins.

In an embodiment, the kit may be adapted to be used in the method ofisolating extracellular vesicles and identifying membrane proteins.

In another embodiment, the kit may be adapted to be used in the methodof diagnosing diseases using tissue or disease-specific membraneproteins on extracellular vesicles from body fluids.

The present disclosure provides an apparatus for implementing the methodof isolating extracellular vesicles and identifying membrane proteinsand the method of diagnosing diseases using tissue or disease-specificmembrane proteins on extracellular vesicles from body fluids.

The present disclosure further provides a computer program productincluding non-transitory computer-readable storage media havingcomputer-readable instructions stored thereon, the computer-readableinstructions being executable by a computerized device comprisingprocessing hardware to execute the method of isolating extracellularvesicles and identifying membrane proteins and the method of diagnosingdiseases using tissue or disease-specific membrane proteins onextracellular vesicles from body fluids, described herein above.

The invention is now described by means of non-limiting examples.

EXAMPLES

In the following, the examples of the present disclosure will bedescribed. The examples concern methods of processing sample material,as well as apparatus that is operable to implement the methods ofprocessing sample material; for example, the apparatus is implemented ina form of a sample processing kit.

Example 1: Presence of Extracellular Vesicles in Interstitial Space of aTumor Tissue Revealed by Electron Microscopy

Materials and Method:

A method developed relates to a study that was approved by the RegionalEthical Review Board at the University of Gothenburg (096-12) and allparticipants provided a written informed consent. The method includesobtaining melanoma metastatic tissues from patients (i.e. participants)during surgery. Thereafter, the method includes preserving the melanomametastatic tissues in complete cell media (without fetal bovine serum)at a temperature of 4° C., until extracellular vesicle isolation isachieved. Thereafter, the method includes placing the samples of thetissue in 150 μm deep membrane carriers (Leica Microsystems) filled with20% BSA in PBS followed by high pressure freezing using EMPactI machine(Leica Microsystems). Thereafter, the method includes applying a freezesubstitution protocol, as follows: 2% uranyl acetate in dehydratedacetone for 1 h. The method includes increasing the temperature by 3° C.per hour up to −50° C. Thereafter, the method includes washing thesamples two times with dehydrated acetone and then infiltrating withincreasing concentrations of HM20 (3:1, 2:1, 1:1, 1:2, 1:3 acetone:HM20)followed with 3 changes with HM20. Thereafter, the method includespolymerizing the samples using UV light for 48 h. Thereafter, the methodincludes cutting thin sections (70 nm) by using a Leica UC6ultramicrotome (Leica Microsystems). Thereafter, the method includescontrasting the sections using 2% uranyl acetate for 4 min and leadcitrate for 2 min.

Results:

The method provides a result, wherein melanin (stained as black) isclearly visible inside melanoma cells; in a practical use of the method,two melanoma cells are well recognized by their cell membrane 100 asshown in FIG. 1. Moreover, in FIG. 1, in the interstitial space oftissues, small size of vesicular structures with various size andmorphology are found. This result shows that the interstitial space isabundant with many extracellular vesicles.

Example 2: Isolation of Extracellular Vesicles by Three DifferentProtocols

Materials and Method:

A method 200 is provided for isolating extracellular vesicles frommelanoma metastatic tissues using centrifugation-based protocols. In themethod 200, three slightly different protocols are beneficiallyemployed, and have been tested in practice. As illustrated in FIG. 2,the method 200 starts at step 202. At the step 202, human tissues areobtained from tumor or diseased tissues.

Protocol-1: Two tumor pieces are treatable by employing the protocol 1,to isolate extracellular vesicles. In the protocol-1, the tumor piecesare gently sliced into small fragments (1-2 mm) 204. At step 206, theplurality of pieces of the human tissue is incubated at 37° C.Thereafter, after a filtration step (70 μm pore size), cells and tissuedebris are eliminated by centrifugation at 300×g for 10 min and 2000×gfor 20 min, as step 210. Supernatant are centrifuged at 16,500×g for 20min, as step 212, and 110,000×g for 2.5 h, as step 214, to collectlarger vesicles and smaller vesicles, respectively; such centrifugationis achieved using, for example, a Ti45 rotor: fixed angle rotor. At step216, all centrifugations are beneficially performed at a temperature of4° C. Moreover, in the protocol 1, pellets are beneficially re-suspendedin PBS.

Protocol-2: After extracellular vesicle isolation is achieved using theprotocol 1, the protocol 2 includes incubating the pellet of larger andsmaller vesicles after final ultracentrifugation with Collagenase D(Roche) (2 mg/ml) and DNase I (Roche) (40 U/ml) dissolved in RPMI plainmedium (Sigma Aldrich) for 30 min at 37° C., as step 220. At step 222and 224, after enzymatic treatments, the protocol 2 includescentrifuging larger vesicles at 16,500×g for 15 min and smaller vesiclesat 117,000×g for 112 min (Ti70 rotor, fixed angle rotor) to removeenzymes and collect the pellets again. Beneficially, all centrifugationsare performed at a temperature of 4° C. Moreover, beneficially, thepellets are re-suspended in PBS.

Protocol-3: As illustrated in FIG. 2, the method 200 may further includestep 218. At the step 2218, the plurality of pieces of the human tissueis incubated at 37° C., or any other temperature, in presence ofcollagenase and DNase. Thereafter, at the step 208 the extracellularvesicles are isolated from the incubated chopped pieces of human tissue.This protocol-3 including gently slicing tumor pieces into smallfragments (1-2 mm) and immediately thereafter incubating the smallfragments with Collagenase D (2 mg/ml) and DNase I (40 U/ml) dissolvedin RPMI plain medium (Sigma Aldrich) for 30 min at 37° C. After afiltration step (70 μm pore size), the protocol 3 includes eliminatingcells and tissue debris by centrifugation at 300×g for 10 min and 2000×gfor 20 min and then collecting larger and smaller vesicles aftercentrifugation at 16,500×g for 20 min and 110,000×g for 2.5 hrespectively; for example, such centrifugation is achieved using a Ti45rotor: fixed angle rotor. Moreover, all centrifugations are beneficiallyperformed at a temperature of 4° C. Resulting pellets are thenre-suspended in PBS.

For negative stain electron microscopy, the protocol 3 includes placinga drop (10 μl) of isolated extracellular vesicles (both larger andsmaller vesicles) onto a glow discharged 200-mesh, namely formvar/carbonCu copper grids for 15 min (Electron Microscopy Sciences, PA, USA) for 5min. Thereafter, the protocol 3 includes washing vesicles three times inPBS and then fixing them using 2% paraformaldehyde. After three furtherwashes in PBS, the protocol 3 includes fixing the samples inglutaraldehyde 2.5%, thereafter washing multiple times (up to fivetimes) in filtered water and staining using 2% uranyl acetate for 5 min.The protocol 3 then includes obtaining pictures using an electronmicroscope, for example a digitized LEO 912AB Omega electron microscope(Carl Zeiss SMT) at 120 kV equipped with a Veleta CCD camera(Olympus-SiS).

Using the aforementioned protocol-1, protocol-2 and protocol-3, it isfeasible to extract RNA from both larger and smaller vesicles isolatedfrom melanoma metastatic tissues using miRCURY™ RNA Isolation Kit(Exiqon) according to an associated manufacturer's protocol. Thereafter,RNA profiles are analyzed using a capillary electrophoresis system (forexample, an Agilent RNA 6000 Nano Kit for larger and smaller vesiclesisolated using protocol 1 and Agilent RNA 6000 Pico Kit for larger andsmaller vesicles isolated using protocols 2 and 3). In the protocols 1,2 and 3, samples are beneficially analyzed using, for example, anAgilent 2100 Bioanalyzer machine (Agilent Technologies).

Results:

Regardless of which of the aforementioned protocols are employed,extracellular vesicles thereby obtained are susceptible to showingtypical RNA profiles and morphology of extracellular vesiclesillustrated in FIGS. 3A to 3F. Larger vesicles that are pelleted at16,500×g are susceptible, in practice, to having small RNAs as well as18S and 28S ribosomal RNA peaks, as illustrated in FIGS. 3A to 3C, upperpanels. This is comparable to a subpopulation of extracellular vesicles,often called microvesicles, which are released by budding of plasmamembrane of cells.

By contrast, smaller vesicles that are pelleted at 110,000×g aresusceptible, in practice, to having small RNAs, but no relativelyprominent ribosomal RNA peaks. This is similar to exosomes, asubpopulation of extracellular vesicles that are released throughendocytic pathway, as illustrated in FIGS. 3D to 3E, upper panels. Inaddition, electron microscopy pictures having been found to show thatlarger vesicles are 100-300 nm in diameter, wherein the typical sizerange of microvesicles, as illustrated in FIGS. 3A to 3C, lower panels,and smaller vesicles are 40 nm to 100 nm in diameter, namely akin to thetypical size range of exosomes, as illustrated in FIGS. 3D to 3E, lowerpanels. In fact, a result employing the aforementioned protocols showsthat enzymatic treatment does not affect molecular characteristics andmorphology of extracellular vesicles.

Example 3: Membrane Isolation and Proteomics Analysis of ExtracellularVesicles

Materials and Method:

In a method pursuant to the present disclosure, extracellular vesiclesfrom two melanoma metastatic tissues (MeT1 and MeT2) are isolated byemploying the aforementioned protocol-2 described in Example 2 above.Additionally, pursuant to the method of Example 3, extracellularvesicles are isolated from three cell lines, MML1 (melanoma cell line),HMC1 (human mast cell line), and MSC (human mesenchymal stem cells).Conditioned media from cell cultures is then harvested and centrifugedat 300×g for 10 min to remove cells. The supernatant is then centrifugedat 2,000×g for 20 min to remove apoptotic bodies and cell debris. Largerand smaller vesicles are then pelleted at 16,500×g for 20 min and at118,000×g (Type 45 Ti, Beckman Coulter) for 3.5 h. Thereafter, isolatedextracellular vesicles are incubated with 100 mM sodium carbonatesolution (pH 12) for 1 h at room temperature with rotation beingemployed. A potassium chloride solution (1 M) is added and furtherincubation for 1 h is then employed. Thereafter, samples hereby derivedare subjected to an OptiPrep density gradient purification, thereafterultracentrifuged at 178,000×g (SW 41 Ti, Beckman Coulter) for 2 h, andthen membranes are collected from an interface between 30% and 10%iodixanol layers associated with such ultracentrifugation.

Thereafter, a proteome of the membrane of extracellular vesicles isidentified by employing LC-MS/MS. Briefly, 30 μg of membranes arebeneficially lysed with 2% SDS and sonicated. Tryptic digestion ofproteins is then beneficially conducted by employing Filter AssociatedAided Sample Preparation. Thereafter, digested peptides are analyzedwith an OrbiTrap mass spectrometer. Peak lists of MS data are generatedfrom the mass spectrometer and peptides/proteins are identified andquantified using a MaxQuant quantification tool with an Andromeda searchengine (version 1.5.2.8). There is thereby obtained quantitative data,namely by way of employing label-free quantification (LFQ) with aminimum of two ratio counts was applied. Moreover, a normalized LFQintensity is thereby obtainable. Moreover, protein localizationinformation is obtainable by employing Uniprot(http://www.uniprot.org/).

Results:

The method of Example 3 is capable of providing example results, as willnext be described. For example, in total, 1239, 901, 1451, 1410, and 959proteins are susceptible to being identified from extracellular vesiclesof MeT1, MeT2, MML1, HMC1, and MSCs, respectively. Only membraneproteins that are annotated in the Uniprot database are beneficiallyselected and compared to determine unique surface protein profiles ofdifferent extracellular vesicles, as illustrated in exemplary graphs602-612 of FIGS. 6A-F, respectively. Sixteen proteins frommelanoma-derived extracellular vesicles (MeT1, MeT2, and MML1) and 92proteins from melanoma metastatic tissue-derived extracellular vesicles(MeT1 and MeT2) are beneficially selected as cancer-specific surfaceproteins, as they were not detected in the other membrane isolates, asshown in van diagram 400 of FIG. 4. In addition, 32 proteins arebeneficially selected among the 133 common proteins, because theseproteins are 4-fold higher in abundance in the melanoma-derivedextracellular vesicles compared to non-melanoma-derived extracellularvesicles, but are highly expressed in the three melanoma-derivedextracellular vesicles, as shown in an exemplary graph 500 of FIG. 5.These results show that melanoma tissue-derived extracellular vesicleshave a unique membrane protein profile, and that embodiments of thepresent disclosure enable such unique membrane protein profiles to bedetected and identified.

Example 4: Detection of Cancer Specific Membrane Proteins from IsolatedExtracellular Vesicles

Materials and Method:

There is provided a method for this Example 4. The method includescoating extracellular vesicles on 96-well plates for an overnight periodat a temperature of 4° C. Thereafter, the method includes removingunbound extracellular vesicles, wherein the plates are blocked with 1%BSA in PBS for 1 h and incubated with anti-COX6c (Santa CruzBiotechnology), anti-SLC25A22 (Santa Cruz Biotechnology), anti-MT-CO2(Abcam), anti-HLA-DR (Santa Cruz Biotechnology), anti-Erlin-2 (Abcam),or anti-RPN1 (Thermo Fisher Scientific) antibodies for 2 h. Afterwashing, the appropriate secondary antibodies with HRP are added.Thereafter, the method includes initiating the reaction by adding a TMBsubstrate solution, terminated by 2M H₂SO₄; the method includesmeasuring the optical density at an interrogating radiation wavelengthof 450 nm.

Results:

For the method of Example 4, results are obtainable, wherein membraneproteins are highly expressed in melanoma metastatic tissue-derivedextracellular vesicles compared to cell line-derived extracellularvesicles, with high correlation of the LFQ intensity from massspectrometry, as illustrated in exemplary graphs 602-612 of FIGS. 6A-F.

Example 5: Development of Sandwich ELISA System to Detect the DoubleMembrane Proteins on Extracellular Vesicles

Materials and Method:

Overall scheme of sandwich ELISA is described in FIG. 7. MT-CO2 antibodywas coated on black 96-well plates for overnight at 4° C. The MT-CO2antibody was purified on a protein G column prior to use to remove thecarrier proteins. The isolated extracellular vesicles were added to thewells and incubated for 2 h at room temperature. After washing with PBS,anti-COX6c, anti-CD9, anti-HLA-DR, or anti-RPN1 antibody was incubatedfor 1 h and then HRP-conjugated anti-mouse antibody was incubated for 1h. Luminescent signal was obtained with the BM Chemiluminescence ELISASubstrate (BD Biosciences).

Results:

High luminescent signals were observed only in melanoma tissue-derivedextracellular vesicles compared with cell line-derived extracellularvesicles when COX6c antibody was used, as shown in an exemplary graph802 of FIG. 8A. Furthermore, when CD9 antibody was used, highluminescent signals were observed, but there was no difference observedbetween samples, as shown in an exemplary graph 804 of FIG. 8B.Moreover, high luminescent signals were missing when HLA-DR or RPN1antibody were used, as shown in exemplary graphs 806, 808 of FIGS. 8C-D.Therefore, based on the FIGS. 8A-D, a conclusion can be made that theextracellular vesicles include different surface molecular profiles, andthe system of the present disclosure works efficiently to detect thecancer specific mitochondrial membrane proteins.

Example 6: Detection of Double Membrane Proteins on ExtracellularVesicles from Patient Samples

Materials and Method:

In the Example 6, there is provided method including steps as will nextbe described. The method includes collecting a total of 20 ml ofperipheral blood from melanoma patients and healthy controls in EDTAtubes. The method next includes a step of obtaining plasma therefrom bycentrifugation at 1880×g for 10 min, followed by a second centrifugationat 2500×g for 10 min. All centrifugations are beneficially performed ata temperature of 4° C.

For example, the method is implemented by collecting blood samples fromovarian cancer patients after anesthesia, but before performing surgeryupon the cancer patients. Beneficially, the method includes collecting 6ml of blood in EDTA vacutainers using standardized procedures, and thencentrifuging and directly aliquoting resulting sample material intoEppendorf tubes, followed by steps of frozing and storing the samplematerial at a temperature of −80° C. within a period in a range of 30minutes to 60 minutes after withdrawal. Moreover, the method includescollecting ovarian cystic fluids after one or more ovarian cysts aresurgically removed and prepared as described above.

In the method, subsequent steps include purifying an MT-CO2 antibodythat is purified by a protein G column prior to use to remove thecarrier proteins, then coating onto black 96-well plates for anovernight period at a temperature of 4° C. Thereafter, the plasma orcystic fluid are added to the wells and incubated for 2 h at roomtemperature. Beneficial, a total of 50 μl of blood plasma from patientsand 30 μl of cystic fluid plasma is used when implementing the method.After washing with PBS, the method includes incubating a COX6c antibodywas incubated for a period of 1 h and then incubating HRP-conjugatedanti-mouse antibody for a period of 1 h. Thereafter, the method includesobtaining a luminescent signal by employing a BM Chemiluminescence ELISASubstrate (BD Biosciences).

Results:

When employing the method of Example 4, higher levels of combined MI-CO2and COX6c are susceptible to being detected in plasma of melanomapatients compared with healthy controls as shown in exemplary graph 902of FIG. 9A with good ROC curve (AUC=0.8684) characteristics as shown inexemplary graph 904 of FIG. 9B. In addition, MT-CO2 and COX6c aresusceptible to being detected in the plasma of both patients with benignand malignant ovarian cysts, without significant differences beingobserved, but the expression level is higher compared with the healthycontrols for both groups as shown in exemplary graph 1002 of FIG. 10Awith good ROC curve (AUC=0.6976) characteristics as shown in exemplarygraph 1004 of FIG. 10B. Importantly, when the MT-CO2/COX6c is quantifiedin ovarian cystic fluids directly, a significant difference ispotentially observed between benign and ovarian cancer cysts as shown inexemplary graph 1102 of FIG. 11A with good ROC curve (AUC=0.9444)characteristics as shown in exemplary graph 1104 of FIG. 11A. Theseresults suggest that cancer specific membrane proteins on extracellularvesicles are released in malignant disease and can be used as validbiomarkers.

Example 7: Detection of the Nucleic Acids Proteins on ExtracellularVesicles

Extracellular vesicles have an aqueous, cargo-containing core surroundedby a roughly spherical bilayer membrane. Arrival of the vesicles at adistant site and fusion with targeted cells allows transport of cargo asdiverse as nucleic acids (DNA, mRNA, and microRNA), proteins, andlipids, facilitating important cell-cell communications.

Materials and Method:

MT-CO2 antibody was coated on black 96-well plates for overnight at 4°C. The MT-CO2 antibody was purified on a protein G column prior to useto remove the membrane proteins. The extraction of the nucleic acids isdone through a spin column-based procedure using affinity membranebinding of all extracellular vesicles. The process of extracting nucleicacids from the isolated extracellular vesicles includes pre-filteringthe sample to exclude cell-contamination, and loading on the membraneaffinity column followed by a brief wash. The bound vesicles are lysedand eluted with QIAzol; the nucleic acid is extracted by addition ofchloroform, precipitated by ethanol and further purified by using anRNeasy column. The extracted nucleic acid is washed with PBS,anti-COX6c, anti-CD9, anti-HLA-DR, or anti-RPN1 antibody and wasincubated for 1 h and then HRP-conjugated anti-mouse antibody wasincubated for 1 h.

Further, to characterize the nucleic acid eluate, 1 μl of the eluate wassubjected to the Bioanalyzer RNA 6000 Pico assay according to themanufacturer's instructions. The aligned fluorescence trace data wasexported from the instrument's software into a CSV file and plottedusing Microsoft Excel.

Results:

High luminescent signals were observed only in melanoma tissue-derivedextracellular vesicles compared with cell line-derived extracellularvesicles when COX6c antibody was used. Furthermore, when CD9 antibodywas used, high luminescent signals were observed, but there was nodifference observed between samples. Moreover, high luminescent signalswere missing when HLA-DR or RPN1 antibody were used. Therefore, based onthe high luminescent signals nature of nucleic acid present on theextracellular vesicles is identified.

Example 8: Detection of Tissue-Specific Membrane Protein by Using AlphaLISA Donor and Acceptor Beads

Materials and Method:

In the Example 8, there is provided method including steps as will nextbe described. The method includes collecting a total of 20 ml ofperipheral blood from melanoma patients in EDTA tubes. The method nextincludes a step of obtaining plasma therefrom by centrifugation at1880×g for 10 min, followed by a second centrifugation at 2500×g for 10min. All centrifugations are beneficially performed at a temperature of4° C.

For example, the method is implemented by collecting blood samples fromovarian cancer patients. Beneficially, the method includes collecting 6ml of blood in EDTA vacutainers using standardized procedures, and thencentrifuging and directly aliquoting resulting sample material intoEppendorf tubes, followed by steps of frozing and storing the samplematerial at a temperature of −80° C. within a period in a range of 30minutes to 60 minutes after withdrawal.

In the method, subsequent steps include purifying an MT-CO2 antibodythat is purified by a protein G column prior to use to remove thecarrier proteins, then coating onto black 96-well plates for anovernight period at a temperature of 4° C. Thereafter, the plasma orcystic fluid are added to the wells and incubated for 2 h at roomtemperature. Beneficial, a total of 50 μl of blood plasma from patientsand 30 μl of cystic fluid plasma is used when implementing the method.After washing with PBS, the method includes incubating a COX6c antibodywas incubated for a period of 1 h and then incubating HRP-conjugatedanti-mouse antibody for a period of 1 h. Thereafter, the method includesobtaining a luminescent signal by employing a BM Chemiluminescence ELISASubstrate (BD Biosciences).

Results:

When employing the method of Example 8, higher luminescent signals wereobserved only in melanoma tissue-derived extracellular vesicles comparedwith cell line-derived extracellular vesicles when COX6c antibody wasused.

Furthermore, when CD9 antibody was used, high luminescent signals wereobserved, but there was no difference observed between samples.Moreover, high luminescent signals were missing when HLA-DR or RPN1antibody were used. Therefore, based on the higher luminescent signals,a conclusion can be made that the extracellular vesicles includedifferent surface molecular profiles, and the system of the presentdisclosure works efficiently to detect the cancer specific mitochondrialmembrane proteins.

Modifications to embodiments described in the foregoing are possiblewithout departing from the scope of the invention as defined by theaccompanying claims. Expressions such as “including”, “comprising”,“incorporating”, “consisting of”, “have”, “is” used to describe andclaim the present invention are intended to be construed in anon-exclusive manner, namely allowing for items, components or elementsnot explicitly described also to be present. Reference to the singularis also to be construed to relate to the plural. Numerals includedwithin parentheses in the accompanying claims are intended to assistunderstanding of the claims and should not be construed in any way tolimit subject matter claimed by these claims.

APPENDIX TO THE SPECIFICATION

TABLE 1 Uniprot ID Protein name Gene name Primary Localization P05023Sodium/potassium-transporting ATP1A1 Cell membrane ATPase subunitalpha-1 Q16891 MICOS complex subunit MIC60 IMMT Mitochondrion innermembrane Q07065 Cytoskeleton-associated protein 4 CKAP4 Endoplasmicreticulum membrane Q13423 NAD(P) transhydrogenase, NNT Mitochondrioninner mitochondrial membrane Q9NVI7 ATPase family AAA domain-containingATAD3A Mitochondrion inner protein 3A membrane P04843Dolichyl-diphosphooligosaccharide-- RPN1 Endoplasmic reticulum proteinglycosyltransferase subunit 1 membrane P25705 ATP synthase subunitalpha, ATP5A1 Mitochondrion inner mitochondrial membrane Q9UJS0Calcium-binding mitochondrial carrier SLC25A13 Mitochondrion innerprotein Aralar2 membrane P27824 Calnexin CANX Endoplasmic reticulummembrane Q9Y4W6 AFG3-like protein 2 AFG3L2 Mitochondrion membrane O60313Dynamin-like 120 kDa protein, OPA1 Mitochondrion inner mitochondrialmembrane P16615 Sarcoplasmic/endoplasmic reticulum ATP2A2 Endoplasmicreticulum calcium ATPase 2 membrane Q5T9A4 ATPase family AAAdomain-containing ATAD3B Mitochondrion inner protein 3B membrane Q6NUK1Calcium-binding mitochondrial carrier SLC25A24 Mitochondrion innerprotein SCaMC-1 membrane O95202 LETM1 and EF-hand domain-containingLETM1 Mitochondrion inner protein 1, mitochondrial membrane P11717Cation-independent mannose-6- IGF2R Lysosome membrane phosphate receptorP21796 Voltage-dependent anion-selective VDAC1 Mitochondrion outerchannel protein 1 membrane O75746 Calcium-binding mitochondrial carrierSLC25A12 Mitochondrion inner protein Aralar1 membrane P50416 CarnitineO-palmitoyltransferase 1, CPT1A Mitochondrion outer liver isoformmembrane Q13724 Mannosyl-oligosaccharide glucosidase MOGS Endoplasmicreticulum membrane Q99623 Prohibitin-2 PHB2 Mitochondrion inner membraneQ07954 Prolow-density lipoprotein receptor- LRP1 Cell membrane relatedprotein 1 Q9UGP8 Translocation protein SEC63 homolog SEC63 Endoplasmicreticulum membrane P02730 Band 3 anion transport protein SLC4A1 Cellmembrane Q00610 Clathrin heavy chain 1 CLTC Cytoplasmic vesicle membraneP54886 Delta-1-pyrroline-5-carboxylate ALDH18A1 Mitochondrion innersynthase membrane P20020 Plasma membrane calcium- ATP2B1 Cell membranetransporting ATPase 1 Q9BWM7 Sideroflexin-3 SFXN3 Mitochondrion membraneQ9HDC9 Adipocyte plasma membrane- APMAP Membrane associated proteinP00387 NADH-cytochrome b5 reductase 3 CYB5R3 Endoplasmic reticulummembrane Q00325 Phosphate carrier protein, SLC25A3 Mitochondrion innermitochondrial membrane P35232 Prohibitin PHB Mitochondrion innermembrane Endoplasmic reticulum-Golgi P49257 Protein ERGIC-53 LMAN1intermediate compartment membrane Q02978 Mitochondrial2-oxoglutarate/malate SLC25A11 Mitochondrion inner carrier proteinmembrane Q9H9B4 Sideroflexin-1 SFXN1 Mitochondrion membrane P46977Dolichyl-diphosphooligosaccharide-- STT3A Endoplasmic reticulum proteinglycosyltransferase subunit membrane STT3A Q8N766 ER membrane proteincomplex EMC1 Membrane subunit 1 O94905 Erlin-2 ERLIN2 Endoplasmicreticulum membrane Q14254 Flotillin-2 FLOT2 Cell membrane P05556Integrin beta-1 ITGB1 Cell membrane Q9NTJ5 Phosphatidylinositidephosphatase SACM1L Endoplasmic reticulum SAC1 membrane Q93084Sarcoplasmic/endoplasmic reticulum ATP2A3 Nucleus membrane calciumATPase 3 P49748 Very long-chain specific acyl-CoA ACADVL Mitochondrioninner dehydrogenase, mitochondrial membrane P05141 ADP/ATP translocase 2SLC25A5 Mitochondrion inner membrane P12236 ADP/ATP translocase 3SLC25A6 Mitochondrion inner membrane P04844Dolichyl-diphosphooligosaccharide- RPN2 Endoplasmic reticulum proteinglycosyltransferase subunit 2 membrane O75477 Erlin-1 ERLIN1 Endoplasmicreticulum membrane O75955 Flotillin-1 FLOT1 Cell membrane Q92896 Golgiapparatus protein 1 GLG1 Golgi apparatus membrane P19367 Hexokinase-1HK1 Mitochondrion outer membrane P30443 HLA class I histocompatibilityantigen, HLA-A Membrane A-1 alpha chain P18463 HLA class Ihistocompatibility antigen, HLA-B Membrane B-37 alpha chain Q969V3Nicalin NCLN Endoplasmic reticulum membrane P23634 Plasma membranecalcium- ATP2B4 Cell membrane transporting ATPase 4 P08575 Receptor-typetyrosine-protein PTPRC Membrane phosphatase C Q15005 Signal peptidasecomplex subunit 2 SPCS2 Microsome membrane Q96PQ0 VPS10domain-containing receptor SORCS2 Membrane SorCS2 P08195 4F2cell-surface antigen heavy chain SLC3A2 Apical cell membrane P32189Glycerol kinase GK Mitochondrion outer membrane P06756 Integrin alpha-VITGAV Membrane Q14165 Malectin MLEC Endoplasmic reticulum membraneQ9HD20 Manganese-transporting ATPase 13A1 ATP13A1 Endoplasmic reticulummembrane Q15155 Nodal modulator 1 NOMO1 Membrane Q93050 V-type protonATPase 116 kDa ATP6V0A1 Cytoplasmic vesicle subunit a isoform 1 membraneQ53H12 Acylglycerol kinase, mitochondrial AGK Mitochondrion membraneP12235 ADP/ATP translocase 1 SLC25A4 Mitochondrion inner membrane Q12797Aspartyl/asparaginyl beta-hydroxylase ASPH Endoplasmic reticulummembrane O75027 ATP-binding cassette sub-family B ABCB7 Mitochondrioninner member 7, mitochondrial membrane P51572 B-cell receptor-associatedprotein 31 BCAP31 Endoplasmic reticulum membrane O75844 CAAX prenylprotease 1 homolog ZMPSTE24 Endoplasmic reticulum membrane Q02127Dihydroorotate dehydrogenase DHODH Mitochondrion inner (quinone),mitochondrial membrane P27105 Erythrocyte band 7 integral STOM Cellmembrane membrane protein P04439 HLA class I histocompatibility antigen,HLA-A Membrane A-3 alpha chain P16189 HLA class I histocompatibilityantigen, HLA-A Membrane A-31 alpha chain P16190 HLA class Ihistocompatibility antigen, HLA-A Membrane A-33 alpha chain Q04826 HLAclass I histocompatibility antigen, HLA-B Membrane B-40 alpha chainP30481 HLA class I histocompatibility antigen, HLA-B Membrane B-44 alphachain P30490 HLA class I histocompatibility antigen, HLA-B Membrane B-52alpha chain P30508 HLA class I histocompatibility antigen, HLA-CMembrane Cw-12 alpha chain Q16853 Membrane primary amine oxidase AOC3Cell membrane Q9NX63 MICOS complex subunit MIC19 CHCHD3 Mitochondrioninner membrane Q9NZM1 Myoferlin MYOF Cell membrane Q9UH99 SUNdomain-containing protein 2 SUN2 Nucleus inner membrane P45880Voltage-dependent anion-selective VDAC2 Mitochondrion outer channelprotein 2 membrane Q96A33 Coiled-coil domain-containing protein CCDC47Membrane 47 P13073 Cytochrome c oxidase subunit 4 COX4I1 Mitochondrioninner isoform 1, mitochondrial membrane P07099 Epoxide hydrolase 1 EPHX1Microsome membrane P10321 HLA class I histocompatibility antigen, HLA-CMembrane Cw-7 alpha chain Q14108 Lysosome membrane protein 2 SCARB2Lysosome membrane O00264 Membrane-associated progesterone PGRMC1Microsome membrane receptor component 1 Q9UBV2 Protein sel-1 homolog 1SEL1L Endoplasmic reticulum membrane Q9UJZ1 Stomatin-like protein 2,mitochondrial STOML2 Mitochondrion inner membrane P02786 Transferrinreceptor protein 1 TFRC Cell membrane O43493 Trans-Golgi networkintegral TGOLN2 Cell membrane membrane protein 2 Q12907 Vesicularintegral-membrane protein LMAN2 Endoplasmic reticulum-Golgi VIP36intermediate compartment membrane P09543 2,3-cyclic-nucleotide 3- CNPMembrane phosphodiesterase Q96ER9 Coiled-coil domain-containing proteinCCDC51 Membrane 51 P08574 Cytochrome c1, heme protein, CYC1Mitochondrion inner mitochondrial membrane P39656Dolichyl-diphosphooligosaccharide-- DDOST Endoplasmic reticulum proteinglycosyltransferase 48 kDa membrane subunit Q8TCJ2Dolichyl-diphosphooligosaccharide-- STT3B Endoplasmic reticulum proteinglycosyltransferase subunit membrane STT3B Q53GQ0 Estradiol17-beta-dehydrogenase 12 HSD17B12 Endoplasmic reticulum membrane P29992Guanine nucleotide-binding protein GNA11 Cell membrane subunit alpha-11P10319 HLA class I histocompatibility antigen, HLA-B Membrane B-58 alphachain P30498 HLA class I histocompatibility antigen, HLA-B Membrane B-78alpha chain P13761 HLA class II histocompatibility HLA-DRB1 Cellmembrane antigen, DRB1-7 beta chain P05107 Integrin beta-2 ITGB2Membrane P05106 Integrin beta-3 ITGB3 Cell membrane Q6UXV4 MICOS complexsubunit MIC27 APOOL Mitochondrion inner membrane Q9Y276 Mitochondrialchaperone BCS1 BCS1L Mitochondrion inner membrane Q9H936 Mitochondrialglutamate carrier 1 SLC25A22 Mitochondrion inner membrane Q6PIU2 Neutralcholesterol ester hydrolase 1 NCEH1 Membrane P61619 Protein transportprotein Sec61 SEC61A1 Endoplasmic reticulum subunit alpha isoform 1membrane O94901 SUN domain-containing protein 1 SUN1 Nucleus innermembrane Q9HD45 Transmembrane 9 superfamily TM9SF3 Membrane member 3Q9NZ01 Very-long-chain enoyl-CoA reductase TECR Endoplasmic reticulummembrane Q9Y277 Voltage-dependent anion-selective VDAC3 Mitochondrionouter channel protein 3 membrane O95870 Abhydrolase domain-containingABHD16A Membrane protein 16A Q8TB61 Adenosine 3-phospho 5- SLC35B2 Golgiapparatus membrane phosphosulfate transporter 1 Q969X5 Endoplasmicreticulum-Golgi ERGIC1 Endoplasmic reticulum intermediate compartmentprotein 1 membrane O94766 Galactosylgalactosylxylosylprotein 3- B3GAT3Golgi apparatus membrane beta-glucuronosyltransferase 3 Q9H3K2 Growthhormone-inducible GHITM Mitochondrion inner transmembrane proteinmembrane P30462 HLA class I histocompatibility antigen, HLA-B MembraneB-14 alpha chain P30466 HLA class I histocompatibility antigen, HLA-BMembrane B-18 alpha chain Q31612 HLA class I histocompatibility antigen,HLA-B Membrane B-73 alpha chain P30501 HLA class I histocompatibilityantigen, HLA-C Membrane Cw-2 alpha chain Q30154 HLA class IIhistocompatibility HLA-DRB5 Cell membrane antigen, DR beta 5 chainP50281 Matrix metalloproteinase-14 MMP14 Membrane P50336Protoporphyrinogen oxidase PPOX Mitochondrion inner membrane Q15907Ras-related protein Rab-llB RAB11B Recycling endosome membrane Q92544Transmembrane 9 superfamily TM9SF4 Membrane member 4 P53007Tricarboxylate transport protein, SLC25A1 Mitochondrion innermitochondrial membrane P21589 5-nucleotidase NT5E Cell membrane Q9NRK6ATP-binding cassette sub-family B ABCB10 Mitochondrion inner member 10,mitochondrial membrane P80723 Brain acid soluble protein 1 BASP1 Cellmembrane O96005 Cleft lip and palate transmembrane CLPTM1 Membraneprotein 1 P22695 Cytochrome b-c1 complex subunit 2, UQCRC2 Mitochondrioninner mitochondrial membrane P09669 Cytochrome c oxidase subunit 6CCOX6C Mitochondrion inner membrane Q7Z2K6 Endoplasmic reticulum ERMP1Endoplasmic reticulum metallopeptidase 1 membrane P42892Endothelin-converting enzyme 1 ECE1 Cell membrane P29317 Ephrin type-Areceptor 2 EPHA2 Cell membrane P16452 Erythrocyte membrane protein bandEPB42 Cell membrane 4.2 P63092 Guanine nucleotide-binding protein GNASCell membrane G(s) subunit alpha isoforms short P01911 HLA class IIhistocompatibility HLA-DRB1 Cell membrane antigen, DRB1-15 beta chainQ08431 Lactadherin MFGE8 Membrane Q9UIQ6 Leucyl-cystinyl aminopeptidaseLNPEP Cell membrane Q9H0U3 Magnesium transporter protein 1 MAGT1 Cellmembrane Q14728 Major facilitator superfamily MFSD10 Membranedomain-containing protein 10 Q8TCT9 Minor histocompatibility antigen H13HM13 Endoplasmic reticulum membrane O43772 Mitochondrialcarnitine/acylcarnitine SLC25A20 Mitochondrion inner carrier proteinmembrane Q9Y6C9 Mitochondrial carrier homolog 2 MTCH2 Mitochondrioninner membrane Q92542 Nicastrin NCSTN Membrane P16671 Plateletglycoprotein 4 CD36 Cell membrane P61006 Ras-related protein Rab-8ARAB8A Cell membrane Q9Y512 Sorting and assembly machinery SAMM50Mitochondrion outer component 50 homolog membrane Q9H3N1Thioredoxin-related transmembrane TMX1 Membrane protein 1 Q99805Transmembrane 9 superfamily TM9SF2 Endosome membrane member 2 Q13445Transmembrane emp24 TMED1 Cell membrane domain-containing protein 1Q7Z7H5 Transmembrane emp24 TMED4 Endoplasmic reticulum domain-containingprotein 4 membrane Q9Y3B3 Transmembrane emp24 TMED7 Endoplasmicreticulum domain-containing protein 7 membrane Q9BVK6 Transmembraneemp24 TMED9 Endoplasmic reticulum domain-containing protein 9 membraneP57088 Transmembrane protein 33 TMEM33 Membrane Q9Y487 V-type protonATPase 116 kDa ATP6V0A2 Cell membrane subunit a isoform 2 Q13488 V-typeproton ATPase 116 kDa TCIRG1 Membrane subunit a isoform 3 Q15904 V-typeproton ATPase subunit S1 ATP6AP1 Vacuole membrane P49641Alpha-mannosidase 2x MAN2A2 Golgi apparatus membrane Q9NW15 Anoctamin-10ANO10 Cell membrane P35613 Basigin BSG Cell membrane Q9UHQ4 B-cellreceptor-associated protein 29 BCAP29 Endoplasmic reticulum membraneQ6KCM7 Calcium-binding mitochondrial carrier SLC25A25 Mitochondrioninner protein SCaMC-2 membrane Q9NPL8 Complex I assembly factor TIMMDC1,TIMMDC1 Mitochondrion membrane mitochondrial O43169 Cytochrome b5 type BCYB5B Mitochondrion outer membrane P31930 Cytochrome b-c1 complexsubunit 1, UQCRC1 Mitochondrion inner mitochondrial membrane Q7KZN9Cytochrome c oxidase assembly COX15 Mitochondrion membrane protein COX15homolog P20674 Cytochrome c oxidase subunit 5A, COX5A Mitochondrioninner mitochondrial membrane P10606 Cytochrome c oxidase subunit 5B,COX5B Mitochondrion inner mitochondrial membrane Q9Y673Dolichyl-phosphate beta- ALG5 Endoplasmic reticulum glucosyltransferasemembrane Q75T13 GPI inositol-deacylase PGAP1 Endoplasmic reticulummembrane Q92643 GPI-anchor transamidase PIGK Endoplasmic reticulummembrane P04233 HLA class II histocompatibility CD74 Cell membraneantigen gamma chain P56199 Integrin alpha-1 ITGA1 Membrane P26006Integrin alpha-3 ITGA3 Cell membrane P11117 Lysosomal acid phosphataseACP2 Lysosome membrane P11279 Lysosome-associated membrane LAMP1 Cellmembrane glycoprotein 1 O15173 Membrane-associated progesterone PGRMC2Membrane receptor component 2 Q13505 Metaxin-1 MTX1 Membrane Q9NZJ7Mitochondrial carrier homolog 1 MTCH1 Mitochondrion inner membraneQ9Y3D7 Mitochondrial import inner membrane PAM16 Mitochondrion innertranslocase subunit TIM16 membrane O96008 Mitochondrial import receptorsubunit TOMM40 Mitochondrion outer TOM40 homolog membrane Q15070Mitochondrial inner membrane OXA1L Mitochondrion inner protein OXA1Lmembrane O96011 Peroxisomal membrane protein 11B PEX11B Peroxisomemembrane Q9HBL7 Plasminogen receptor (KT) PLGRKT Cell membrane P16284Platelet endothelial cell adhesion PECAM1 Cell membrane molecule Q16647Prostacyclin synthase PTGIS Endoplasmic reticulum membrane O15258Protein RER1 RER1 Golgi apparatus membrane P61026 Ras-related proteinRab-10 RAB10 Cytoplasmic vesicle membrane P51148 Ras-related proteinRab-5C RAB5C Cell membrane P10301 Ras-related protein R-Ras RRAS Cellmembrane Q8TC12 Retinol dehydrogenase 11 RDH11 Endoplasmic reticulummembrane P67812 Signal peptidase complex catalytic SEC11A Microsomemembrane subunit SEC11A Q9Y5M8 Signal recognition particle receptorSRPRB Endoplasmic reticulum subunit beta membrane P54709Sodium/potassium-transporting ATP1B3 Cell membrane ATPase subunit beta-3P11166 Solute carrier family 2, facilitated SLC2A1 Cell membrane glucosetransporter member 1 P31040 Succinate dehydrogenase SDHA Mitochondrioninner [ubiquinone] flavoprotein subunit, membrane mitochondrial O00161Synaptosomal-associated protein 23 SNAP23 Cell membrane Q99442Translocation protein SEC62 SEC62 Endoplasmic reticulum membrane P51571Translocon-associated protein subunit SSR4 Endoplasmic reticulum deltamembrane Q6ZXV5 Transmembrane and TPR repeat- TMTC3 Membrane containingprotein 3 Q15363 Transmembrane emp24 TMED2 Cytoplasmic vesicledomain-containing protein 2 membrane Q9BVC6 Transmembrane protein 109TMEM109 Nucleus outer membrane Q6UW68 Transmembrane protein 205 TMEM205Membrane O75396 Vesicle-trafficking protein SEC22b SEC22B Endoplasmicreticulum membrane Q8TDW0 Volume-regulated anion channel LRRC8C Cellmembrane subunit LRRC8C Q6PML9 Zinc transporter 9 SLC30A9 MembraneQ9NUQ2 1-acyl-sn-glycerol-3-phosphate AGPAT5 Endoplasmic reticulumacyltransferase epsilon membrane Q06136 3-ketodihydrosphingosinereductase KDSR Endoplasmic reticulum membrane O00400 Acetyl-coenzyme Atransporter 1 SLC33A1 Endoplasmic reticulum membrane P26572Alpha-1,3-mannosyl-glycoprotein 2- MGAT1 Golgi apparatus membranebeta-N-acetylglucosaminyltransferase Q10469Alpha-1,6-mannosyl-glycoprotein 2- MGAT2 Golgi apparatus membranebeta-N-acetylglucosaminyltransferase Q8NE86 Calcium uniporter protein,MCU Mitochondrion inner mitochondrial membrane P16070 CD44 antigen CD44Cell membrane P21926 CD9 antigen CD9 Membrane Q9NZ45 CDGSH iron-sulfurdomain-containing CISD1 Mitochondrion outer protein 1 membrane Q8N5K1CDGSH iron-sulfur domain-containing CISD2 Endoplasmic reticulum protein2 membrane Q9NV96 Cell cycle control protein 50A TMEM30A Membrane P43121Cell surface glycoprotein MUC18 MCAM Membrane Q96G23 Ceramide synthase 2CERS2 Nucleus membrane Q96S66 Chloride channel CLIC-like protein 1 CLCC1Membrane Q96KA5 Cleft lip and palate transmembrane CLPTM1L Membraneprotein 1-like protein O94886 CSC1-like protein 1 TMEM63A Lysosomemembrane P13498 Cytochrome b-245 light chain CYBA Cell membrane Q6IAN0Dehydrogenase/reductase SDR family DHRS7B Endoplasmic reticulum member7B membrane O94923 D-glucuronyl C5-epimerase GLCE Golgi apparatusmembrane P61803 Dolichyl-diphosphooligosaccharide- DAD1 Endoplasmicreticulum protein glycosyltransferase subunit membrane DADI P49961Ectonucleoside triphosphate ENTPD1 Membrane diphosphohydrolase 1 Q9P0I2ER membrane protein complex EMC3 Membrane subunit 3 Q9NPA0 ER membraneprotein complex EMC7 Membrane subunit 7 A0FGR8 Extended synaptotagmin-2ESYT2 Cell membrane P36269 Gamma-glutamyltransferase 5 GGT5 MembraneP50440 Glycine amidinotransferase, GATM Mitochondrion innermitochondrial membrane Q96S52 GPI transamidase component PIG-S PIGSEndoplasmic reticulum membrane Q969N2 GPI transamidase component PIG-TPIGT Endoplasmic reticulum membrane P63244 Guanine nucleotide-bindingprotein GNB2L1 Cell membrane subunit beta-2-like 1 P30273 High affinityimmunoglobulin epsilon FCER1G Cell membrane receptor subunit gammaP13747 HLA class I histocompatibility antigen, HLA-E Membrane alphachain E P01903 HLA class II histocompatibility HLA-DRA Cell membraneantigen, DR alpha chain Q70UQ0 Inhibitor of nuclear factor kappa-B IKBIPEndoplasmic reticulum kinase-interacting protein membrane P17301Integrin alpha-2 ITGA2 Membrane P23229 Integrin alpha-6 ITGA6 Cellmembrane P05362 Intercellular adhesion molecule 1 ICAM1 Membrane Q6UWP7Lysocardiolipin acyltransferase 1 LCLAT1 Endoplasmic reticulum membraneQ9BQT8 Mitochondrial 2-oxodicarboxylate SLC25A21 Mitochondrion innercarrier membrane Q9UBX3 Mitochondrial dicarboxylate carrier SLC25A10Mitochondrion inner membrane Q96DA6 Mitochondrial import inner membraneDNAJC19 Mitochondrion inner translocase subunit TIM14 membrane Q969M1Mitochondrial import receptor subunit TOMM40L Mitochondrion outer TOM40Bmembrane Q9Y619 Mitochondrial ornithine transporter 1 SLC25A15Mitochondrion inner membrane P26038 Moesin MSN Cell membrane Q8N2K0Monoacylglycerol lipase ABHD12 ABHD12 Membrane Q9P0J0 NADH dehydrogenase[ubiquinone] 1 NDUFA13 Mitochondrion inner alpha subcomplex subunit 13membrane Q9UHQ9 NADH-cytochrome b5 reductase 1 CYB5R1 Membrane O15118Niemann-Pick C1 protein NPC1 Late endosome membrane Q9UQ90 ParapleginSPG7 Mitochondrion membrane O60568 Procollagen-lysine,2-oxoglutarate 5-PLOD3 Rough endoplasmic dioxygenase 3 reticulum membrane Q9HCJ1Progressive ankylosis protein ANKH Membrane homolog P23219 ProstaglandinG/H synthase 1 PTGS1 Microsome membrane Q96JJ7 Proteindisulfide-isomerase TMX3 TMX3 Endoplasmic reticulum membrane Q96A26Protein FAM162A FAM162A Membrane Q96ND0 Protein FAM210A FAM210A MembraneQ9H0X4 Protein ITFG3 ITFG3 Membrane Q96AA3 Protein RFT1 homolog RFT1Membrane P63000 Ras-related C3 botulinum toxin RAC1 Cell membranesubstrate 1 P61020 Ras-related protein Rab-5B RAB5B Cell membrane P11233Ras-related protein Ral-A RALA Cell surface P62834 Ras-related proteinRap-1A RAP1A Cell membrane P61224 Ras-related protein Rap-1b RAP1B Cellmembrane P61225 Ras-related protein Rap-2b RAP2B Recycling endosomemembrane O75787 Renin receptor ATP6AP2 Membrane Q9NY15 Stabilin-1 STAB1Membrane Q9BX79 Stimulated by retinoic acid gene 6 STRA6 Cell membraneprotein homolog Q86WV6 Stimulator of interferon genes protein TMEM173Endoplasmic reticulum membrane Q15526 Surfeit locus protein 1 SURF1Mitochondrion inner membrane O15400 Syntaxin-7 STX7 Early endosomemembrane O15533 Tapasin TAPBP Endoplasmic reticulum membrane Q9Y320Thioredoxin-related transmembrane TMX2 Membrane protein 2 P24557Thromboxane-A synthase TBXAS1 Endoplasmic reticulum membrane Q5JTV8Torsin-1A-interacting protein 1 TOR1AIP Nucleus inner membrane 1 Q15629Translocating chain-associated TRAM1 Endoplasmic reticulum membraneprotein 1 membrane Q9H061 Transmembrane protein 126A TMEM126AMitochondrion inner membrane Q6PI78 Transmembrane protein 65 TMEM65Membrane Q9BQA9 Uncharacterized protein C17orf62 C17orf62 MembraneQ96GQ5 UPF0420 protein C16orf58 C16orf58 Membrane Q9P035 Very-long-chain(3R)-3-hydroxyacyl- PTPLAD1 Endoplasmic reticulum CoA dehydratase 3membrane A1L0T0 Acetolactate synthase-like protein ILVBL Membrane P28907ADP-ribosyl cyclase/cyclic ADP-ribose CD38 Membrane hydrolase 1 P21397Amine oxidase [flavin-containing] A MAOA Mitochondrion outer membraneP27338 Amine oxidase [flavin-containing] B MAOB Mitochondrion outermembrane O95782 AP-2 complex subunit alpha-1 AP2A1 Cell membrane O94973AP-2 complex subunit alpha-2 AP2A2 Cell membrane Q13557Calcium/calmodulin-dependent CAMK2D Cell membrane protein kinase type IIsubunit delta P20645 Cation-dependent mannose-6- M6PR Lysosome membranephosphate receptor Q5ZPR3 CD276 antigen CD276 Membrane P08962 CD63antigen CD63 Cell membrane P48960 CD97 antigen CD97 Cell membrane O14735CDP-diacylglycerol-inositol 3- CDIPT Endoplasmic reticulumphosphatidyltransferase membrane Q9BT22 Chitobiosyldiphosphodolicholbeta- ALG1 Endoplasmic reticulum mannosyltransferase membrane Q5T3F8CSC1-like protein 2 TMEM63B Membrane Q9GZY4 Cytochrome c oxidaseassembly COA1 Mitochondrion inner factor 1 homolog membrane Q5RI15Cytochrome c oxidase protein 20 COX20 Mitochondrion membrane homologP00403 Cytochrome c oxidase subunit 2 MT-CO2 Mitochondrion innermembrane O14672 Disintegrin and metalloproteinase ADAM10 Cell membranedomain-containing protein 10 Q9H3Z4 DnaJ homolog subfamily C member 5DNAJC5 Membrane Q9NX47 E3 ubiquitin-protein ligase MARCH5 MARCH5Mitochondrion outer membrane Q96K19 E3 ubiquitin-protein ligase RNF170RNF170 Endoplasmic reticulum membrane Q86TM6 E3 ubiquitin-protein ligasesynoviolin SYVN1 Endoplasmic reticulum membrane Q9UKM7 Endoplasmicreticulum mannosyl- MAN1B1 Endoplasmic reticulum oligosaccharide1,2-alpha- membrane mannosidase Q96RQ1 Endoplasmic reticulum-GolgiERGIC2 Endoplasmic reticulum-Golgi intermediate compartment protein 2intermediate compartment membrane Q9Y282 Endoplasmic reticulum-GolgiERGIC3 Endoplasmic reticulum-Golgi intermediate compartment protein 3intermediate compartment membrane Q5J8M3 ER membrane protein complexEMC4 Membrane subunit 4 O75063 Glycosaminoglycan xylosylkinase FAM20BGolgi apparatus membrane Q5H8A4 GPI ethanolamine phosphate PIGGEndoplasmic reticulum transferase 2 membrane P59768 Guaninenucleotide-binding protein GNG2 Cell membrane G(I)/G(S)/G(O) subunitgamma-2 P12314 High affinity immunoglobulin gamma FCGR1A Cell membraneFc receptor I P04440 HLA class II histocompatibility HLA-DPB1 Cellmembrane antigen, DP beta 1 chain P01920 HLA class II histocompatibilityHLA-DQB1 Cell membrane antigen, DQ beta 1 chain Q14573 Inositol1,4,5-trisphosphate receptor ITPR3 Endoplasmic reticulum type 3 membraneP08069 Insulin-like growth factor 1 receptor IGF1R Cell membrane P20702Integrin alpha-X ITGAX Membrane P32942 Intercellular adhesion molecule 3ICAM3 Membrane Q8NC56 LEM domain-containing protein 2 LEMD2 Nucleusinner membrane Q96AG4 Leucine-rich repeat-containing protein LRRC59Microsome membrane 59 Q08722 Leukocyte surface antigen CD47 CD47 Cellmembrane Q6PCB7 Long-chain fatty acid transport SLC27A1 Cell membraneprotein 1 P33121 Long-chain-fatty-acid--CoA ligase 1 ACSL1 Mitochondrionouter membrane O14880 Microsomal glutathione S-transferase MGST3Endoplasmic reticulum 3 membrane Q9H2D1 Mitochondrial folate SLC25A3Mitochondrion inner transporter/carrier 2 membrane Q9BVV7 Mitochondrialimport inner membrane TIMM21 Mitochondrion membrane translocase subunitTim21 O14925 Mitochondrial import inner membrane TIMM23 Mitochondrioninner translocase subunit Tim23 membrane O95563 Mitochondrial pyruvatecarrier 2 MPC2 Mitochondrion inner membrane P53985 Monocarboxylatetransporter 1 SLC16A1 Cell membrane P08571 Monocyte differentiationantigen CD14 Cell membrane CD14 O96000 NADH dehydrogenase [ubiquinone] 1NDUFB10 Mitochondrion inner beta subcomplex subunit 10 membrane O43674NADH dehydrogenase [ubiquinone] 1 NDUFB5 Mitochondrion inner betasubcomplex subunit 5, membrane mitochondrial O95169 NADH dehydrogenase[ubiquinone] 1 NDUFB8 Mitochondrion inner beta subcomplex subunit 8,membrane mitochondrial Q9Y6M9 NADH dehydrogenase [ubiquinone] 1 NDUFB9Mitochondrion inner beta subcomplex subunit 9 membrane O95298 NADHdehydrogenase [ubiquinone] 1 NDUFC2 Mitochondrion inner subunit C2membrane O75306 NADH dehydrogenase [ubiquinone] NDUFS2 Mitochondrioninner iron-sulfur protein 2, mitochondrial membrane O75489 NADHdehydrogenase [ubiquinone] NDUFS3 Mitochondrion inner iron-sulfurprotein 3, mitochondrial membrane Q8IY17 Neuropathy target esterasePNPLA6 Endoplasmic reticulum membrane Q9Y639 Neuroplastin NPTN Cellmembrane Q8IXM6 Nurim NRM Nucleus inner membrane Q86WC4Osteopetrosis-associated OSTM1 Lysosome membrane transmembrane protein 1Q9NYL4 Peptidyl-prolyl cis-trans isomerase FKBP11 Membrane FKBP11 O75915PRA1 family protein 3 ARL6IP5 Endoplasmic reticulum membrane Q6P4E1Protein CASC4 CASC4 Membrane Q8N5M9 Protein jagunal homolog 1 JAGN1Endoplasmic reticulum membrane P60468 Protein transport protein Sec61SEC61B Endoplasmic reticulum subunit beta membrane P51153 Ras-relatedprotein Rab-13 RAB13 Cell membrane Q15286 Ras-related protein Rab-35RAB35 Cell membrane P20339 Ras-related protein Rab-5A RAB5A Cellmembrane Q92930 Ras-related protein Rab-8B RAB8B Cell membrane Q00765Receptor expression-enhancing REEP5 Membrane protein 5 Q16799Reticulon-1 RTN1 Endoplasmic reticulum membrane Q9NQC3 Reticulon-4 RTN4Endoplasmic reticulum membrane Q9NRX5 Serine incorporator 1 SERINCIEndoplasmic reticulum membrane O15269 Serine palmitoyltransferase 1SPTLC1 Endoplasmic reticulum membrane Q96HS1 Serine/threonine-proteinphosphatase PGAM5 Mitochondrion outer PGAM5, mitochondrial membraneQ6P4A7 Sideroflexin-4 SFXN4 Mitochondrion inner membrane Q9BY50 Signalpeptidase complex catalytic SEC11C Microsome membrane subunit SEC11CQ9Y6A9 Signal peptidase complex subunit 1 SPCS1 Microsome membraneP61009 Signal peptidase complex subunit 3 SPCS3 Microsome membraneP08240 Signal recognition particle receptor SRPR Endoplasmic reticulumsubunit alpha membrane P05026 Sodium/potassium-transporting ATP1B1 Cellmembrane ATPase subunit beta-1 Q9BSK2 Solute carrier family 25 member 33SLC25A33 Mitochondrion inner membrane Q8TBP6 Solute carrier family 25member 40 SLC25A40 Mitochondrion inner membrane Q96H78 Solute carrierfamily 25 member 44 SLC25A44 Mitochondrion inner membrane Q12846Syntaxin-4 STX4 Cell membrane Q9H1E5 Thioredoxin-related transmembraneTMX4 Membrane protein 4 P43307 Translocon-associated protein subunitSSR1 Endoplasmic reticulum alpha membrane O15321 Transmembrane 9superfamily TM9SF1 Lysosome membrane member 1 Q9Y3A6 Transmembrane emp24domain- TMED5 Endoplasmic reticulum containing protein 5 membrane P17152Transmembrane protein 11, TMEM11 Mitochondrion inner mitochondrialmembrane Q9NX00 Transmembrane protein 160 TMEM160 Membrane Q9UHN6Transmembrane protein 2 TMEM2 Membrane Q9H330 Transmembrane protein 245TMEM245 Membrane Q9H3H5 UDP-N-acetylglucosamine--dolichyl- DPAGT1Endoplasmic reticulum phosphate N- membraneacetylglucosaminephosphotransferase Q96IX5 Up-regulated during skeletalmuscle USMG5 Mitochondrion membrane growth protein 5 Q15836Vesicle-associated membrane protein 3 VAMP3 Membrane Q9P0L0Vesicle-associated membrane VAPA Endoplasmic reticulumprotein-associated protein A membrane Q999431-acyl-sn-glycerol-3-phosphate AGPAT1 Endoplasmic reticulumacyltransferase alpha membrane Q15125 3-beta-hydroxysteroid- EBPEndoplasmic reticulum Delta(8),Delta(7)-isomerase membrane Q9UBM77-dehydrocholesterol reductase DHCR7 Endoplasmic reticulum membraneQ15041 ADP-ribosylation factor-like protein 6- ARL6IP1 Endomembranesystem interacting protein 1 Q9NVJ2 ADP-ribosylation factor-like proteinARL8B Late endosome membrane 8B Q9H6U8 Alpha-1,2-mannosyltransferaseALG9 ALG9 Endoplasmic reticulum membrane P15144 Aminopeptidase N ANPEPCell membrane Q4KMQ2 Anoctamin-6 ANO6 Cell membrane Q03518 Antigenpeptide transporter 1 TAPI Endoplasmic reticulum membrane Q9BUR5Apolipoprotein O APOO Membrane P20292 Arachidonate 5-lipoxygenase-ALOX5AP Nucleus membrane activating protein Q6DD88 Atlastin-3 ATL3Endoplasmic reticulum membrane P00846 ATP synthase subunit a MT-ATP6Mitochondrion inner membrane O94911 ATP-binding cassette sub-family AABCA8 Cell membrane member 8 P28288 ATP-binding cassette sub-family DABCD3 Peroxisome membrane member 3 Q16611 Bcl-2 homologousantagonist/killer BAK1 Mitochondrion membrane Q9Y6X5Bis(5-adenosyl)-triphosphatase ENPP4 Cell membrane ENPP4 P11836B-lymphocyte antigen CD20 MS4A1 Cell membrane P55290 Cadherin-13 CDH13Cell membrane Q8NCH0 Carbohydrate sulfotransferase 14 CHST14 Golgiapparatus membrane Q9ULX7 Carbonic anhydrase 14 CA14 Membrane P26678Cardiac phospholamban PLN Sarcoplasmic reticulum membrane P23786Carnitine O-palmitoyltransferase 2, CPT2 Mitochondrion innermitochondrial membrane Q03135 Caveolin-1 CAV1 Golgi apparatus membraneQ9HA82 Ceramide synthase 4 CERS4 Nucleus membrane Q8N5B7 Ceramidesynthase 5 CERS5 Nucleus membrane Q9Y6K0Choline/ethanolaminephosphotransferase 1 CEPT1 Endoplasmic reticulummembrane Q6UVK1 Chondroitin sulfate proteoglycan 4 CSPG4 Apical cellmembrane Q96HD1 Cysteine-rich with EGF-like domain CRELD1 Membraneprotein 1 Q53TN4 Cytochrome b reductase 1 CYBRD1 Membrane P04839Cytochrome b-245 heavy chain CYBB Cell membrane O14949 Cytochrome b-c1complex subunit 8 UQCRQ Mitochondrion inner membrane Q9UDW1 Cytochromeb-c1 complex subunit 9 UQCR10 Mitochondrion inner membrane Q9Y2R0Cytochrome c oxidase assembly COA3 Mitochondrion inner factor 3 homolog,mitochondrial membrane O00483 Cytochrome c oxidase subunit NDUFA4Mitochondrion inner NDUFA4 membrane Q6UW02 Cytochrome P450 20A1 CYP20A1Membrane Q6UVY6 DBH-like monooxygenase protein 1 MOXD1 Endoplasmicreticulum membrane P78536 Disintegrin and metalloproteinase ADAM17Membrane domain-containing protein 17 Q9NXW2 DnaJ homolog subfamily Bmember DNAJB12 Membrane 12 Q9Y5T4 DnaJ homolog subfamily C memberDNAJC15 Mitochondrion inner 15 membrane Q9Y672 Dolichyl pyrophosphateMan9GlcNAc2 ALG6 Endoplasmic reticulum alpha-1,3-glucosyltransferasemembrane Q9BV10 Dol-P-Man:Man(7)GlcNAc(2)-PP-Dol ALG12 Endoplasmicreticulum alpha-1,6-mannosyltransferase membrane P17813 Endoglin ENGMembrane Q99808 Equilibrative nucleoside transporter 1 SLC29A1Basolateral cell membrane Q5UCC4 ER membrane protein complex EMC10Membrane subunit 10 O43909 Exostosin-like 3 EXTL3 Endoplasmic reticulummembrane P15311 Ezrin EZR Apical cell membrane P51648 Fatty aldehydedehydrogenase ALDH3A2 Endoplasmic reticulum membrane Q9Y4F1 FERM, RhoGEFand pleckstrin FARP1 Cell membrane domain-containing protein 1 Q9NXS2Glutaminyl-peptide cyclotransferase- QPCTL Golgi apparatus membrane likeprotein P02724 Glycophorin-A GYPA Cell membrane O43292 Glycosylphosphatidylinositol anchor GPAA1 Endoplasmic reticulum attachment 1protein membrane Q7Z5G4 Golgin subfamily A member 7 GOLGA7 Golgiapparatus membrane Q9UBI6 Guanine nucleotide-binding protein GNG12 Cellmembrane G(I)/G(S)/G(O) subunit gamma-12 P51798 H(+)/Cl(−) exchangetransporter 7 CLCN7 Lysosome membrane P20036 HLA class IIhistocompatibility HLA-DPA1 Cell membrane antigen, DP alpha 1 chainP55899 IgG receptor FcRn large subunit p51 FCGRT Cell membrane Q14571Inositol 1,4,5-trisphosphate receptor ITPR2 Endoplasmic reticulum type 2membrane P08648 Integrin alpha-5 ITGA5 Membrane P08514 Integrinalpha-IIb ITGA2B Membrane P18084 Integrin beta-5 ITGB5 Membrane P13164Interferon-induced transmembrane IFITM1 Cell membrane protein 1 Q9NPH3Interleukin-1 receptor accessory IL1RAP Cell membrane protein Q9Y624Junctional adhesion molecule A F11R Cell junction Q9BX67 Junctionaladhesion molecule C JAM3 Cell membrane Q5SVS4 Kidney mitochondrialcarrier protein 1 SLC25A30 Mitochondrion inner membrane Q14739 Lamin-Breceptor LBR Nucleus inner membrane Q6P1Q0 LETM1 domain-containingprotein 1 LETMD1 Mitochondrion outer membrane Q9BU23 Lipase maturationfactor 2 LMF2 Endoplasmic reticulum membrane Q6P1M0 Long-chain fattyacid transport SLC27A4 Membrane protein 4 P12318 Low affinityimmunoglobulin gamma FCGR2A Cell membrane Fc region receptor II-a P19256Lymphocyte function-associated CD58 Cell membrane antigen 3 Q6P1A2Lysophospholipid acyltransferase 5 LPCAT3 Endoplasmic reticulum membraneP21757 Macrophage scavenger receptor types MSR1 Membrane I and II O75352Mannose-P-dolichol utilization defect MPDU1 Membrane 1 protein Q4ZIN3Membralin TMEM259 Membrane Q5TGZ0 MICOS complex subunit MIC10 MINOS1Mitochondrion inner membrane Q8N8R3 Mitochondrial basic amino acidsSLC25A29 Mitochondrion inner transporter membrane Q9GZY8 Mitochondrialfission factor MFF Mitochondrion outer membrane Q9UDX5 Mitochondrialfission process protein 1 MTFP1 Mitochondrion inner membrane O43615Mitochondrial import inner membrane TIMM44 Mitochondrion innertranslocase subunit TIM44 membrane Q3ZCQ8 Mitochondrial import innermembrane TIMM50 Mitochondrion inner translocase subunit TIM50 membraneQ9NS69 Mitochondrial import receptor subunit TOMM22 Mitochondrion outerTOM22 homolog membrane Q8N4H5 Mitochondrial import receptor subunitTOMM5 Mitochondrion outer TOM5 homolog membrane Q96B49 Mitochondrialimport receptor subunit TOMM6 Mitochondrion outer TOM6 homolog membraneQ9Y5U8 Mitochondrial pyruvate carrier 1 MPC1 Mitochondrion innermembrane Q86Y39 NADH dehydrogenase [ubiquinone] 1 NDUFA11 Mitochondrioninner alpha subcomplex subunit 11 membrane O75438 NADH dehydrogenase[ubiquinone] 1 NDUFB1 Mitochondrion inner beta subcomplex subunit 1membrane O43676 NADH dehydrogenase [ubiquinone] 1 NDUFB3 Mitochondrioninner beta subcomplex subunit 3 membrane O95168 NADH dehydrogenase[ubiquinone] 1 NDUFB4 Mitochondrion inner beta subcomplex subunit 4membrane P49821 NADH dehydrogenase [ubiquinone] NDUFV1 Mitochondrioninner flavoprotein 1, mitochondrial membrane P28331 NADH-ubiquinoneoxidoreductase 75 NDUFS1 Mitochondrion inner kDa subunit, mitochondrialmembrane O95674 Phosphatidate cytidylyltransferase 2 CDS2 Mitochondrioninner membrane P48426 Phosphatidylinositol 5-phosphate 4- PIP4K2A Cellmembrane kinase type-2 alpha Q9H490 Phosphatidylinositol glycan anchorPIGU Endoplasmic reticulum biosynthesis class U protein membrane Q2PZI1Probable C-mannosyltransferase DPY19L1 Membrane DPY19L1 Q6ZPD9 ProbableC-mannosyltransferase DPY19L3 Membrane DPY19L3 Q8TED1 Probableglutathione peroxidase 8 GPX8 Membrane Q96KR6 Protein FAM210B FAM210BMembrane Q86UE4 Protein LYRIC MTDH Endoplasmic reticulum membrane P60059Protein transport protein Sec61 SEC61G Endoplasmic reticulum subunitgamma membrane Q5T9L3 Protein wntless homolog WLS Golgi apparatusmembrane Q9GZM5 Protein YIPF3 YIPF3 Cell membrane P35241 Radixin RDXCell membrane Q9NP72 Ras-related protein Rab-18 RAB18 Cell membraneEndoplasmic reticulum-Golgi P61019 Ras-related protein Rab-2A RAB2Aintermediate compartment membrane Q9NRW1 Ras-related protein Rab-6BRAB6B membrane Golgi apparatus membrane P11234 Ras-related protein Ral-BRALB Cell membrane P62070 Ras-related protein R-Ras2 RRAS2 Cell membraneO75298 Reticulon-2 RTN2 Endoplasmic reticulum membrane O95197Reticulon-3 RTN3 Endoplasmic reticulum membrane P84095 Rho-relatedGTP-binding protein RHOG Cell membrane RhoG Q99720 Sigma non-opioidintracellular SIGMAR1 Nucleus inner membrane receptor 1 Q08357Sodium-dependent phosphate SLC20A2 Cell membrane transporter 2 Q9UP95Solute carrier family 12 member 4 SLC12A4 Membrane P11169 Solute carrierfamily 2, facilitated SLC2A3 Membrane glucose transporter member 3O15260 Surfeit locus protein 4 SURF4 Endoplasmic reticulum membraneO43760 Synaptogyrin-2 SYNGR2 Membrane Q16563 Synaptophysin-like protein1 SYPL1 Cytoplasmic vesicle membrane P04216 Thy-1 membrane glycoproteinTHY1 Cell membrane P61586 Transforming protein RhoA RHOA Cell membraneP30536 Translocator protein TSPO Mitochondrion membrane Q9UM00Transmembrane and coiled-coil TMCO1 Endoplasmic reticulumdomain-containing protein 1 membrane Q6NXT6 Transmembrane anteriorposterior TAPT1 Membrane transformation protein 1 homolog Q14956Transmembrane glycoprotein NMB GPNMB Cell membrane Q9BXJ8 Transmembraneprotein 120A TMEM120A Membrane Q53S58 Transmembrane protein 177 TMEM177Membrane Q5SNT2 Transmembrane protein 201 TMEM201 Nucleus inner membraneQ9BUB7 Transmembrane protein 70, TMEM70 Mitochondrion innermitochondrial membrane Q8NBN3 Transmembrane protein 87A TMEM87A MembraneQ9Y275 Tumor necrosis factor ligand TNFSF13B Cell membrane superfamilymember 13B Q13454 Tumor suppressor candidate 3 TUSC3 Endoplasmicreticulum membrane Q9NVA1 Ubiquinol-cytochrome-c reductase UQCC1Mitochondrion inner complex assembly factor 1 membrane Q99807 Ubiquinonebiosynthesis protein COQ7 Mitochondrion inner COQ7 homolog membraneQ9Y385 Ubiquitin-conjugating enzyme E2 J1 UBE2J1 Endoplasmic reticulummembrane Q96F25 UDP-N-acetylglucosamine transferase ALG14 Endoplasmicreticulum subunit ALG14 homolog membrane B0I1T2 Unconventional myosin-IgMYO1G Cell membrane Q6Y1H2 Very-long-chain (3R)-3-hydroxyacyl- PTPLBEndoplasmic reticulum CoA dehydratase 2 membrane Q9UEU0 Vesicletransport through interaction VTI1B Late endosome membrane with t-SNAREshomolog 1B Q9BV40 Vesicle-associated membrane protein VAMP8 Lysosomemembrane 8 Q9H0V9 VIP36-like protein LMAN2L Endoplasmic reticulummembrane Q96D96 Voltage-gated hydrogen channel 1 HVCN1 Membrane P61421V-type proton ATPase subunit d 1 ATP6V0D1 Membrane Q92504 Zinctransporter SLC39A7 SLC39A7 Endoplasmic reticulum membrane Q92604Acyl-CoA: lysophosphatidylglycerol LPGAT1 Endoplasmic reticulumacyltransferase 1 membrane Q01518 Adenylyl cyclase-associated protein 1CAP1 Cell membrane P40616 ADP-ribosylation factor-like protein 1 ARL1Golgi apparatus membrane Q96BM9 ADP-ribosylation factor-like proteinARL8A Late endosome membrane 8A Q16706 Alpha-mannosidase 2 MAN2A1 Golgiapparatus membrane Q02094 Ammonium transporter Rh type A RHAG MembraneQ9Y679 Ancient ubiquitous protein 1 AUP1 Endoplasmic reticulum membraneQ9HCE9 Anoctamin-8 ANO8 Cell membrane P63010 AP-2 complex subunit betaAP2B1 Cell membrane Q96CW1 AP-2 complex subunit mu AP2M1 Cell membraneQ07812 Apoptosis regulator BAX BAX Mitochondrion membrane P10415Apoptosis regulator Bcl-2 BCL2 Mitochondrion outer membrane P29972Aquaporin-1 AQP1 Cell membrane P03928 ATP synthase protein 8 MT-ATP8Mitochondrion membrane Q9BXK5 Bcl-2-like protein 13 BCL2L13Mitochondrion membrane Q8WY22 BRI3-binding protein BRI3BP Mitochondrionouter membrane P08311 Cathepsin G CTSG Cell surface P48509 CD151 antigenCD151 Membrane P13987 CD59 glycoprotein CD59 Cell membrane P60033 CD81antigen CD81 Membrane P27701 CD82 antigen CD82 Membrane P14209 CD99antigen CD99 Membrane P60953 Cell division control protein 42 CDC42 Cellmembrane homolog Q6ZMG9 Ceramide synthase 6 CERS6 Nucleus membraneQ8WWI5 Choline transporter-like protein 1 SLC44A1 Cell membrane Q8IWA5Choline transporter-like protein 2 SLC44A2 Membrane Q96FZ5 CKLF-likeMARVEL transmembrane CMTM7 Membrane domain-containing protein 7 P08174Complement decay-accelerating CD55 Cell membrane factor Q9UBG0 C-typemannose receptor 2 MRC2 Membrane O14569 Cytochrome b561domain-containing CYB561D2 Membrane protein 2 P00414 Cytochrome coxidase subunit 3 MT-CO3 Mitochondrion inner membrane P15954 Cytochromec oxidase subunit 7C, COX7C Mitochondrion inner mitochondrial membraneP10176 Cytochrome c oxidase subunit 8A, COX8A Mitochondrion innermitochondrial membrane Q9BUN8 Derlin-1 DERL1 Endoplasmic reticulummembrane Q9GZP9 Derlin-2 DERL2 Endoplasmic reticulum membrane P15924Desmoplakin DSP Cell junction Q86YN1 Dolichyldiphosphatase 1 DOLPP1Endoplasmic reticulum membrane P22413 Ectonucleotide ENPP1 Cell membranepyrophosphatase/phosphodiesterase family member 1 Q9H4M9 EHdomain-containing protein 1 EHD1 Recycling endosome membrane Q16134Electron transfer flavoprotein- ETFDH Mitochondrion inner ubiquinoneoxidoreductase, membrane mitochondrial P50402 Emerin EMD Nucleus innermembrane P24530 Endothelin B receptor EDNRB Cell membrane Q9BV81 ERmembrane protein complex EMC6 Membrane subunit 6 Q9H4I9 Essential MCUregulator, SMDT1 Mitochondrion inner mitochondrial membrane Q14802 FXYDdomain-containing ion transport FXYD3 Membrane regulator 3 Q96BI3Gamma-secretase subunit APH-1A APH1A Endoplasmic reticulum membraneQ96A29 GDP-fucose transporter 1 SLC35C1 Golgi apparatus membrane O43826Glucose-6-phosphate translocase SLC37A4 Endoplasmic reticulum membraneP04921 Glycophorin-C GYPC Cell membrane Q2TAP0 Golgin subfamily A member7B GOLGA7B Golgi apparatus membrane Q9H3S5 GPI mannosyltransferase 1PIGM Endoplasmic reticulum membrane P63218 Guanine nucleotide-bindingprotein GNG5 Cell membrane G(I)/G(S)/G(O) subunit gamma-5 Q14344 Guaninenucleotide-binding protein GNA13 Membrane subunit alpha-13 Q7LGA3Heparan sulfate 2-O-sulfotransferase HS2ST1 Golgi apparatus membrane 1Q30201 Hereditary hemochromatosis protein HFE Membrane P52789Hexokinase-2 HK2 Mitochondrion outer membrane Q9Y241 HIG1 domain familymember 1A, HIGD1A Mitochondrion membrane mitochondrial P30825 Highaffinity cationic amino acid SLC7A1 Cell membrane transporter 1 P17693HLA class I histocompatibility antigen, HLA-G Membrane alpha chain GP28068 HLA class II histocompatibility HLA-DMB Late endosome membraneantigen, DM beta chain P13765 HLA class II histocompatibility HLA-DOBEndosome membrane antigen, DO beta chain Q96CC6 Inactive rhomboidprotein 1 RHBDF1 Endoplasmic reticulum membrane Q9Y2U8 Inner nuclearmembrane protein LEMD3 Nucleus inner membrane Man1 Q86V85 Integralmembrane protein GPR180 GPR180 Membrane Q9UKX5 Integrin alpha-11 ITGA11Membrane P18564 Integrin beta-6 ITGB6 Membrane Q8N6L1Keratinocyte-associated protein 2 KRTCAP2 Endoplasmic reticulum membraneQ8NC54 Keratinocyte-associated KCT2 Membrane transmembrane protein 2Q9ULH0 Kinase D-interacting substrate of KIDINS220 Membrane 220 kDaQ16850 Lanosterol 14-alpha demethylase CYP51A1 Endoplasmic reticulummembrane P19397 Leukocyte surface antigen CD53 CD53 Cell membrane Q13449Limbic system-associated membrane LSAMP Cell membrane protein Q96S06Lipase maturation factor 1 LMF1 Endoplasmic reticulum membrane O95573Long-chain-fatty-acid-CoA ligase 3 ACSL3 Mitochondrion outer membraneQ92633 Lysophosphatidic acid receptor 1 LPAR1 Cell surface Q8NF37Lysophosphatidylcholine LPCAT1 Endoplasmic reticulum acyltransferase 1membrane Q7L5N7 Lysophosphatidylcholine LPCAT2 Endoplasmic reticulumacyltransferase 2 membrane Q6ZNC8 Lysophospholipid acyltransferase 1MBOAT1 Membrane Q96N66 Lysophospholipid acyltransferase 7 MBOAT7Membrane P13473 Lysosome-associated membrane LAMP2 Cell membraneglycoprotein 2 P84157 Matrix-remodeling-associated protein MXRA7Membrane 7 Q5JRA6 Melanoma inhibitory activity protein 3 MIA3Endoplasmic reticulum membrane Q8TBP5 Membrane protein FAM174A FAM174AMembrane Q5EB52 Mesoderm-specific transcript homolog MEST Endoplasmicreticulum protein membrane Q99735 Microsomal glutathione S-transferaseMGST2 Endoplasmic reticulum 2 membrane Q9Y584 Mitochondrial import innermembrane TIMM22 Mitochondrion inner translocase subunit Tim22 membraneO94826 Mitochondrial import receptor TOMM70A Mitochondrion outer subunitTOM70 membrane Q96LU5 Mitochondrial inner membrane IMMP1L Mitochondrioninner protease subunit 1 membrane Q9BV23 Monoacylglycerol lipase ABHD6ABHD6 Membrane O95297 Myelin protein zero-like protein 1 MPZL1 MembraneQ96S97 Myeloid-associated differentiation MYADM Membrane marker O43505N-acetyllactosaminide beta-1,3-N- B3GNT1 Golgi apparatus membraneacetylglucosaminyltransferase P03905 NADH-ubiquinone oxidoreductaseMT-ND4 Mitochondrion membrane chain 4 P03915 NADH-ubiquinoneoxidoreductase MT-ND5 Mitochondrion inner chain 5 membrane O14786Neuropilin-1 NRP1 Cell membrane Q15758 Neutral amino acid transporterB(0) SLC1A5 Cell membrane Q8N9A8 Nuclear envelope phosphatase- CNEP1R1Nucleus membrane regulatory subunit 1 Q92621 Nuclear pore complexprotein Nup205 NUP205 Nucleus membrane Q9Y2C4 Nuclease EXOG,mitochondrial EXOG Mitochondrion inner membrane Q9P0S3 ORM1-like protein1 ORMDL1 Endoplasmic reticulum membrane Q96BW9 Phosphatidatecytidylyltransferase, TAMM41 Mitochondrion inner mitochondrial membraneQ9BVG9 Phosphatidylserine synthase 2 PTDSS2 Endoplasmic reticulummembrane Q8IV08 Phospholipase D3 PLD3 Endoplasmic reticulum membraneQ9NRY6 Phospholipid scramblase 3 PLSCR3 Mitochondrion membrane P13224Platelet glycoprotein Ib beta chain GP1BB Membrane P09619Platelet-derived growth factor PDGFRB Cell membrane receptor beta O00592Podocalyxin PODXL Apical cell membrane P15151 Poliovirus receptor PVRCell membrane Q8N2U9 PQ-loop repeat-containing protein 1 PQLC1 MembraneO60831 PRA1 family protein 2 PRAF2 Endosome membrane P49810 Presenilin-2PSEN2 Endoplasmic reticulum membrane Q9UKR5 Probable ergosterolbiosynthetic C14orf1 Endoplasmic reticulum protein 28 membrane Q02809Procollagen-lysine,2-oxoglutarate 5- PLOD1 Rough endoplasmic dioxygenase1 reticulum membrane Q9Y284 Protein Asterix WDR83OS Membrane Q9P003Protein cornichon homolog 4 CNIH4 Membrane Q9UBU6 Protein FAM8A1 FAM8A1Membrane Q9C0E8 Protein lunapark LNP Endoplasmic reticulum membraneQ9NQG1 Protein MANBAL MANBAL Membrane P06703 Protein S100-A6 S100A6Nucleus envelope Q8TF72 Protein Shroom3 SHROOM3 Cell junction Q9H2V7Protein spinster homolog 1 SPNS1 Mitochondrion inner membrane Q14761Protein tyrosine phosphatase PTPRCAP Membrane receptor type C-associatedprotein O95070 Protein YIF1A YIF1A Endoplasmic reticulum membrane Q5BJH7Protein YIF1B YIF1B Membrane Q969M3 Protein YIPF5 YIPF5 Endoplasmicreticulum membrane Q04941 Proteolipid protein 2 PLP2 Membrane A6NKF9Putative Golgi pH regulator C GPR89C Membrane Q14699 Raftlin RFTN1 Cellmembrane P46940 Ras GTPase-activating-like protein IQGAP1 Cell membraneIQGAP1 Q9UL25 Ras-related protein Rab-21 RAB21 Endoplasmic reticulummembrane P51159 Ras-related protein Rab-27A RAB27A Membrane Q12913Receptor-type tyrosine-protein PTPRJ Cell membrane phosphatase etaQ969E2 Secretory carrier-associated SCAMP4 Membrane membrane protein 4Q15165 Serum paraoxonase/arylesterase 2 PON2 Membrane A6NMB1 Sialicacid-binding Ig-like lectin SIGLEC16 Membrane 16 P0DJ93 Small integralmembrane protein 13 SMIM13 Membrane Q8N5G0 Small integral membraneprotein 20 SMIM20 Membrane Q8WUM9 Sodium-dependent phosphate SLC20A1Membrane transporter 1 Q9BXP2 Solute carrier family 12 member 9 SLC12A9Cell membrane Q96BI1 Solute carrier family 22 member 18 SLC22A18 Apicalcell membrane Q96GZ6 Solute carrier family 41 member 3 SLC41A3 Cellmembrane Q8NBI5 Solute carrier family 43 member 3 SLC43A3 MembraneO94956 Solute carrier organic anion SLCO2B1 Cell membrane transporterfamily member 2B1 O95136 Sphingosine 1-phosphate receptor 2 S1PR2 Cellmembrane Q15738 Sterol-4-alpha-carboxylate 3- NSDHL Endoplasmicreticulum dehydrogenase, decarboxylating membrane P08842Steryl-sulfatase STS Endoplasmic reticulum membrane O43759Synaptogyrin-1 SYNGR1 Membrane P34741 Syndecan-2 SDC2 MembraneEndoplasmic reticulum-Golgi Q13190 Syntaxin-5 STX5 intermediatecompartment membrane Q9UNK0 Syntaxin-8 STX8 Membrane O00560 Syntenin-1SDCBP Cell junction O60637 Tetraspanin-3 TSPAN3 Membrane Q8NFQ8Torsin-1A-interacting protein 2 TOR1AIP2 Endoplasmic reticulum membraneQ9UNL2 Translocon-associated protein SSR3 Endoplasmic reticulum subunitgamma membrane Q9Y3Q3 Transmembrane emp24 domain- TMED3 Endoplasmicreticulum-Golgi containing protein 3 intermediate compartment membraneQ4V9L6 Transmembrane protein 119 TMEM119 Membrane Q9P0S9 Transmembraneprotein 14C TMEM14C Mitochondrion membrane Q9HC07 Transmembrane protein165 TMEM165 Golgi apparatus membrane Q9P2C4 Transmembrane protein 181TMEM181 Membrane Q6NUQ4 Transmembrane protein 214 TMEM214 Endoplasmicreticulum membrane Q8N2U0 Transmembrane protein 256 TMEM256 MembraneP61165 Transmembrane protein 258 TMEM258 Membrane Q5BJF2 Transmembraneprotein 97 TMEM97 Nucleus membrane Q6ZT21 Transmembrane protein withTMPPE Membrane metallophosphoesterase domain Q8IXB3 Tumor suppressorcandidate 5 TUSC5 Membrane Q9ULQ1 Two pore calcium channel protein 1TPCN1 Lysosome membrane Q8N4L2 Type 2 phosphatidylinositol 4,5- TMEM55ALate endosome membrane bisphosphate 4-phosphatase P07948Tyrosine-protein kinase Lyn LYN Cell membrane Q969S0 UDP-xylose andUDP-N- SLC35B4 Golgi apparatus membrane acetylglucosamine transporterQ8IYS2 Uncharacterized protein KIAA2013 KIAA2013 Membrane Q13336 Ureatransporter 1 SLC14A1 Cell membrane Q3ZAQ7 Vacuolar ATPase assemblyintegral VMA21 Endoplasmic reticulum membrane protein VMA21 membraneQ96GC9 Vacuole membrane protein 1 VMP1 Endoplasmic reticulum-Golgiintermediate compartment membrane Q9Y3E0 Vesicle transport protein GOT1BGOLT1B Golgi apparatus membrane Q8N0U8 Vitamin K epoxide reductasecomplex VKORC1L1 Endoplasmic reticulum subunit 1-like protein 1 membraneP54289 Voltage-dependent calcium channel CACNA2D1 Membrane subunitalpha-2/delta-1 Q8WY21 VPS10 domain-containing receptor SORCS1 MembraneSorCS1 P27449 V-type proton ATPase 16 kDa ATP6V0C Vacuole membraneproteolipid subunit Q15043 Zinc transporter ZIP14 SLC39A14 Cell membraneP08865 40S ribosomal protein SA RPSA Cell membrane P176435,6-dihydroxyindole-2-carboxylic TYRP1 Melanosome membrane acid oxidaseQ00013 55 kDa erythrocyte membrane MPP1 Membrane protein O00767 Acyl-CoAdesaturase SCD Endoplasmic reticulum membrane P25054 Adenomatouspolyposis coli protein APC Cell junction Q10588 ADP-ribosylcyclase/cyclic ADP-ribose BST1 Cell membrane hydrolase 2 P36405ADP-ribosylation factor-like protein 3 ARL3 Golgi apparatus membraneP05186 Alkaline phosphatase, tissue- ALPL Cell membrane nonspecificisozyme O00116 Alkyldihydroxyacetonephosphate AGPS Peroxisome membranesynthase, peroxisomal Q9NVD7 Alpha-parvin PARVA Cell junction P54920Alpha-soluble NSF attachment protein NAPA Membrane P04920 Anion exchangeprotein 2 SLC4A2 Membrane Q86XL3 Ankyrin repeat and LEM domain- ANKLE2Endoplasmic reticulum containing protein 2 membrane O75843 AP-1 complexsubunit gamma-like 2 AP1G2 Golgi apparatus membrane P53680 AP-2 complexsubunit sigma AP2S1 Cell membrane P33897 ATP-binding cassette sub-familyD ABCD1 Peroxisome membrane member 1 P50895 Basal cell adhesion moleculeBCAM Membrane Q13884 Beta-1-syntrophin SNTB1 Cell membrane Q13425Beta-2-syntrophin SNTB2 Membrane Q9HBI1 Beta-parvin PARVB Cell junctionQ9Y5Z0 Beta-secretase 2 BACE2 Membrane Q5VW32 BRO1 domain-containingprotein BROX Membrane BROX Q96CX2 BTB/POZ domain-containing proteinKCTD12 Cell junction KCTD12 Q16581 C3a anaphylatoxin chemotactic C3AR1Cell membrane receptor P55287 Cadherin-11 CDH11 Cell membrane P19022Cadherin-2 CDH2 Cell membrane P10644 cAMP-dependent protein kinase typePRKAR1A Cell membrane I-alpha regulatory subunit P22748 Carbonicanhydrase 4 CA4 Cell membrane P14384 Carboxypeptidase M CPM Cellmembrane P13688 Carcinoembryonic antigen-related cell CEACAM1 Cellmembrane adhesion molecule 1 P40199 Carcinoembryonic antigen-relatedcell CEACAM6 Cell membrane adhesion molecule 6 P31997 Carcinoembryonicantigen-related cell CEACAM8 Cell membrane adhesion molecule 8 Q6YHK3CD109 antigen CD109 Cell membrane Q13740 CD166 antigen ALCAM MembraneO43633 Charged multivesicular body protein CHMP2A Late endosome membrane2a Q96FZ7 Charged multivesicular body protein 6 CHMP6 Endomembranesystem Q9NY35 Claudin domain-containing protein 1 CLDND1 Membrane Q9UGN4CMRF35-like molecule 8 CD300A Cell membrane Q9ULV4 Coronin-1C CORO1CCell membrane P57737 Coronin-7 CORO7 Golgi apparatus membrane P78310Coxsackievirus and adenovirus CXADR Cell membrane receptor Q9H1C7Cysteine-rich and transmembrane CYSTM1 Membrane domain-containingprotein 1 P14927 Cytochrome b-c1 complex subunit 7 UQCRB Mitochondrioninner membrane P47985 Cytochrome b-c1 complex subunit UQCRFS1Mitochondrion inner Rieske, mitochondrial membrane O14548 Cytochrome coxidase subunit 7A- COX7A2L Mitochondrion inner related protein,mitochondrial membrane Q99418 Cytohesin-2 CYTH2 Cell membrane Q9UKG1DCC-interacting protein 13-alpha APPL1 Early endosome membrane Q92608Dedicator of cytokinesis protein 2 DOCK2 Endomembrane system Q15392Delta(24)-sterol reductase DHCR24 Endoplasmic reticulum membrane Q92629Delta-sarcoglycan SGCD Cell membrane Q02413 Desmoglein-1 DSG1 Cellmembrane Q16832 Discoidin domain-containing receptor DDR2 Cell membrane2 Q96PD2 Discoidin, CUB and LCCL domain- DCBLD2 Membrane containingprotein 2 Q12959 Disks large homolog 1 DLG1 Membrane P31689 DnaJ homologsubfamily A member 1 DNAJA1 Membrane O60884 DnaJ homolog subfamily Amember 2 DNAJA2 Membrane Q9P2X0 Dolichol-phosphate DPM3 Endoplasmicreticulum mannosyltransferase subunit 3 membrane P11532 Dystrophin DMDCell membrane Q5T4S7 E3 ubiquitin-protein ligase UBR4 UBR4 MembraneQ9NZN4 EH domain-containing protein 2 EHD2 Cell membrane Q9NZN3 EHdomain-containing protein 3 EHD3 Cell membrane Q9H223 EHdomain-containing protein 4 EHD4 Cell membrane Q9NZ08 Endoplasmicreticulum ERAP1 Endoplasmic reticulum aminopeptidase 1 membrane Q9HCU0Endosialin CD248 Membrane P29320 Ephrin type-A receptor 3 EPHA3 Cellmembrane P54760 Ephrin type-B receptor 4 EPHB4 Cell membrane Q9UBC2Epidermal growth factor receptor EPS15L1 Cell membrane substrate 15-like1 Q96HE7 ERO1-like protein alpha ERO1L Endoplasmic reticulum membraneQ9BSJ8 Extended synaptotagmin-1 ESYT1 Endoplasmic reticulum membraneQ8N6M3 Fat storage-inducing transmembrane FITM2 Endoplasmic reticulumprotein 2 membrane O95864 Fatty acid desaturase 2 FADS2 Endoplasmicreticulum membrane Q9Y5Y0 Feline leukemia virus subgroup C FLVCR1 Cellmembrane receptor-related protein 1 P41440 Folate transporter 1 SLC19A1Membrane Q7Z2K8 G protein-regulated inducer of GPRIN1 Cell membraneneurite outgrowth 1 Q8TB36 Ganglioside-induced differentiation- GDAP1Mitochondrion outer associated protein 1 membrane P17302 Gap junctionalpha-1 protein GJA1 Cell membrane P36383 Gap junction gamma-1 proteinGJC1 Cell membrane P35052 Glypican-1 GPC1 Cell membrane O75487Glypican-4 GPC4 Cell membrane Q9H4G4 Golgi-associated plantpathogenesis- GLIPR2 Golgi apparatus membrane related protein 1 Q9Y653G-protein coupled receptor 56 GPR56 Cell membrane Q9NZH0 G-proteincoupled receptor family C GPRC5B Cell membrane group 5 member B Q9NQ84G-protein coupled receptor family C GPRC5C Cell membrane group 5 memberC P01112 GTPase HRas HRAS Cell membrane P01111 GTPase NRas NRAS Cellmembrane Q14C86 GTPase-activating protein and VPS9 GAPVD1 Membranedomain-containing protein 1 Q15382 GTP-binding protein Rheb RHEBEndomembrane system Q9Y6B6 GTP-binding protein SAR1b SAR1B Endoplasmicreticulum membrane O15431 High affinity copper uptake protein 1 SLC31A1Cell membrane Q5DX21 Immunoglobulin superfamily member IGSF11 Cellmembrane 11 O75054 Immunoglobulin superfamily member IGSF3 Membrane 3Q969P0 Immunoglobulin superfamily member IGSF8 Cell membrane 8 Q71H61Immunoglobulin-like domain- ILDR2 Endoplasmic reticulum containingreceptor 2 membrane Q13308 Inactive tyrosine-protein kinase 7 PTK7Membrane Q9Y287 Integral membrane protein 2B ITM2B Golgi apparatusmembrane P13612 Integrin alpha-4 ITGA4 Membrane Q13683 Integrin alpha-7ITGA7 Membrane P53708 Integrin alpha-8 ITGA8 Membrane P20701 Integrinalpha-L ITGAL Membrane P11215 Integrin alpha-M ITGAM Membrane Q13418Integrin-linked protein kinase ILK Cell junction O15554 Intermediateconductance calcium- KCNN4 Membrane activated potassium channel protein4 P14923 Junction plakoglobin JUP Cell junction Q96J84 Kin of IRRE-likeprotein 1 KIRREL Cell membrane P42167 Lamina-associated polypeptide 2,TMPO Nucleus inner membrane isoforms beta/gamma P20700 Lamin-B1 LMNB1Nucleus inner membrane P40126 L-dopachrome tautomerase DCT Melanosomemembrane Q15334 Lethal(2) giant larvae protein LLGL1 Early endosomemembrane homolog 1 Q14392 Leucine-rich repeat-containing LRRC32 Membraneprotein 32 P11049 Leukocyte antigen CD37 CD37 Membrane P16150Leukosialin SPN Membrane Q16873 Leukotriene C4 synthase LTC4S Nucleusouter membrane Q08477 Leukotriene-B(4) omega-hydroxylase CYP4F3Endoplasmic reticulum 2 membrane P48059 LIM and senescent cellantigen-like- LIMSI Cell junction containing domain protein 1 O43561Linker for activation of T-cells LAT Cell membrane family member 1O14494 Lipid phosphate phosphohydrolase 1 PPAP2A Cell membrane Q6ZUX7Lipoma HMGIC fusion partner-like 2 LHFPL2 Membrane protein Q5K4L6Long-chain fatty acid transport SLC27A3 Mitochondrion membrane protein 3O60488 Long-chain-fatty-acid--CoA ligase 4 ACSL4 Mitochondrion outermembrane P01130 Low-density lipoprotein receptor LDLR Cell membraneQ7Z4W1 L-xylulose reductase DCXR Membrane Q13571 Lysosomal-associatedLAPTM5 Lysosome membrane transmembrane protein 5 Q9NZW5 MAGUK p55subfamily member 6 MPP6 Membrane Q6NUT3 Major facilitator superfamilyMFSD12 Membrane domain-containing protein 12 P04156 Major prion proteinPRNP Cell membrane Q9BSK0 MARVEL domain-containing protein 1 MARVELD1Cell membrane Q8IX19 Mast cell-expressed membrane MCEMP1 Membraneprotein 1 P10721 Mast/stem cell growth factor KIT Cell membrane receptorKit P40967 Melanocyte protein PMEL PMEL Endoplasmic reticulum membraneQ16655 Melanoma antigen recognized by T- MLANA Endoplasmic reticulumcells 1 membrane P08582 Melanotransferrin MFI2 Cell membrane Q658P3Metalloreductase STEAP3 STEAP3 Endosome membrane Q8N3F8 MICAL-likeprotein 1 MICALL1 Recycling endosome membrane Q99595 Mitochondrialimport inner membrane TIMM17A Mitochondrion inner translocase subunitTiml7-A membrane Q15388 Mitochondrial import receptor TOMM20Mitochondrion outer subunit TOM20 homolog membrane Q8IXI1 MitochondrialRho GTPase 2 RHOT2 Mitochondrion outer membrane O15427 Monocarboxylatetransporter 4 SLC16A3 Cell membrane O15403 Monocarboxylate transporter 7SLC16A6 Cell membrane P33527 Multidrug resistance-associated ABCC1 Cellmembrane protein 1 O15439 Multidrug resistance-associated ABCC4 Membraneprotein 4 O75970 Multiple PDZ domain protein MPDZ Cell membrane P60201Myelin proteolipid protein PLP1 Cell membrane Q9UI09 NADH dehydrogenase[ubiquinone] 1 NDUFA12 Mitochondrion inner alpha subcomplex subunit 12membrane P16435 NADPH-cytochrome P450 reductase POR Endoplasmicreticulum membrane Q9Y2A7 Nck-associated protein 1 NCKAP1 Cell membraneP55160 Nck-associated protein 1-like NCKAP1L Cell membrane Q92692Nectin-2 PVRL2 Cell membrane P08473 Neprilysin MME Cell membrane Q8NF91Nesprin-1 SYNE1 Nucleus outer membrane P13591 Neural cell adhesionmolecule 1 NCAM1 Cell membrane O15394 Neural cell adhesion molecule 2NCAM2 Cell membrane Q04721 Neurogenic locus notch homolog NOTCH2 Cellmembrane protein 2 Q9UM47 Neurogenic locus notch homolog NOTCH3 Cellmembrane protein 3 Q7Z3B1 Neuronal growth regulator 1 NEGRI Cellmembrane O60462 Neuropilin-2 NRP2 Membrane P43007 Neutral amino acidtransporter A SLC1A4 Membrane Q8N1F7 Nuclear pore complex protein Nup93NUP93 Nucleus membrane Q99571 P2X purinoceptor 4 P2RX4 Membrane Q9C0B5Palmitoyltransferase ZDHHC5 ZDHHC5 Cell membrane O75781 Paralemmin-1PALM Cell membrane Q96HC4 PDZ and LIM domain protein 5 PDLIM5 Celljunction Q14318 Peptidyl-prolyl cis-trans isomerase FKBP8 Mitochondrionmembrane FKBP8 Q99755 Phosphatidylinositol 4-phosphate 5- PIP5K1A Cellmembrane kinase type-1 alpha O60331 Phosphatidylinositol 4-phosphate 5-PIP5K1C Cell membrane kinase type-1 gamma O15162 Phospholipid scramblase1 PLSCR1 Cell membrane Q9H8W4 Pleckstrin homology domain- PLEKHF2 Earlyendosome membrane containing family F member 2 Q9UIW2 Plexin-A1 PLXNA1Cell membrane O15031 Plexin-B2 PLXNB2 Cell membrane Q9Y4D7 Plexin-D1PLXND1 Cell membrane Q6NZI2 Polymerase I and transcript release PTRFMembrane factor Q9H7F0 Probable cation-transporting ATPase ATP13A3Membrane 13A3 O75340 Programmed cell death protein 6 PDCD6 Nucleusmembrane Q9P2B2 Prostaglandin F2 receptor negative PTGFRN Endoplasmicreticulum regulator membrane Q8TCG1 Protein CIP2A KIAA1524 MembraneO60610 Protein diaphanous homolog 1 DIAPH1 Cell membrane Q99497 ProteinDJ-1 PARK7 Cell membrane Q14156 Protein EFR3 homolog A EFR3A Cellmembrane Q9H8M9 Protein eva-1 homolog A EVA1A Endoplasmic reticulummembrane Q8N5C1 Protein FAM26E FAM26E Membrane Q96RT1 Protein LAP2ERBB2IP Cell junction Q969X1 Protein lifeguard 3 TMBIM1 Membrane Q9NUP9Protein lin-7 homolog C LIN7C Cell membrane Q14160 Protein scribblehomolog SCRIB Cell membrane Q15437 Protein transport protein Sec23BSEC23B Golgi apparatus membrane Q9BSA4 Protein tweety homolog 2 TTYH2Cell membrane Q9C0H2 Protein tweety homolog 3 TTYH3 Cell membrane O75695Protein XRP2 RP2 Cell membrane Q96EC8 Protein YIPF6 YIPF6 MembraneQ14517 Protocadherin Fat 1 FAT1 Cell membrane O60245 Protocadherin-7PCDH7 Cell membrane Q14242 P-selectin glycoprotein ligand 1 SELPLGMembrane A6NGU5 Putative gamma- GGT3P Membrane glutamyltranspeptidase 3Q14644 Ras GTPase-activating protein 3 RASA3 Cell membrane Q9UL26Ras-related protein Rab-22A RAB22A Endosome membrane Q9ULC3 Ras-relatedprotein Rab-23 RAB23 Cell membrane O00194 Ras-related protein Rab-27BRAB27B Membrane Q9H082 Ras-related protein Rab-33B RAB33B Golgiapparatus membrane P57729 Ras-related protein Rab-38 RAB38 Cell membraneQ9NP90 Ras-related protein Rab-9B RAB9B Cell membrane Q16827Receptor-type tyrosine-protein PTPRO Membrane phosphatase 0 Q8IUW5RELT-like protein 1 RELL1 Cell membrane Q8NFJ5 Retinoic acid-inducedprotein 3 GPRC5A Cell membrane Q9Y6N7 Roundabout homolog 1 ROBO1Membrane Q8WTV0 Scavenger receptor class B member 1 SCARB1 Cell membraneO14828 Secretory carrier-associated SCAMP3 Membrane membrane protein 3O94804 Serine/threonine-protein kinase 10 STK10 Cell membrane Q7KZI7Serine/threonine-protein kinase MARK2 Cell membrane MARK2 Q08209Serine/threonine-protein phosphatase PPP3CA Cell membrane 2B catalyticsubunit alpha isoform Q9P0V3 SH3 domain-binding protein 4 SH3BP4Membrane O43699 Sialic acid-binding Ig-like lectin 6 SIGLEC6 Cellmembrane Q9NYZ4 Sialic acid-binding Ig-like lectin 8 SIGLEC8 MembraneQ9UIB8 SLAM family member 5 CD84 Cell membrane O75044 SLIT-ROBO RhoGTPase-activating SRGAP2 Cell membrane protein 2 Q8NHG7 SmallVCP/p97-interacting protein SVIP Smooth endoplasmic reticulum membraneP31641 Sodium- and chloride-dependent SLC6A6 Cell membrane taurinetransporter Q9Y6M7 Sodium bicarbonate cotransporter 3 SLC4A7 Basolateralcell membrane P19634 Sodium/hydrogen exchanger 1 SLC9A1 Membrane Q96QD8Sodium-coupled neutral amino acid SLC38A2 Cell membrane transporter 2Q9Y289 Sodium-dependent multivitamin SLC5A6 Membrane transporter P55011Solute carrier family 12 member 2 SLC12A2 Membrane Q9UGH3 Solute carrierfamily 23 member 2 SLC23A2 Cell membrane Q96K37 Solute carrier family 35member E1 SLC35E1 Membrane Q00796 Sorbitol dehydrogenase SORDMitochondrion membrane Q99523 Sortilin SORT1 Membrane Q13596 Sortingnexin-1 SNX1 Endosome membrane Q96RF0 Sorting nexin-18 SNX18Endomembrane system O60749 Sorting nexin-2 SNX2 Early endosome membraneQ96L92 Sorting nexin-27 SNX27 Early endosome membrane Q9Y5X1 Sortingnexin-9 SNX9 Cytoplasmic vesicle membrane Q02318 Sterol 26-hydroxylase,mitochondrial CYP27A1 Mitochondrion membrane P50443 Sulfate transporterSLC26A2 Membrane O95425 Supervillin SVIL Cell membrane Q86Y82Syntaxin-12 STX12 Endosome membrane Q13277 Syntaxin-3 STX3 MembraneQ9HBL0 Tensin-1 TNS1 Cell surface Q68CZ2 Tensin-3 TNS3 Cell junctionQ8NG11 Tetraspanin-14 TSPAN14 Membrane O14817 Tetraspanin-4 TSPAN4Membrane O60779 Thiamine transporter 1 SLC19A2 Membrane Q07157 Tightjunction protein ZO-1 TJP1 Cell membrane Q9UP52 Transferrin receptorprotein 2 TFR2 Cell membrane Q9Y5S1 Transient receptor potential cationTRPV2 Cell membrane channel subfamily V member 2 P30408 Transmembrane 4L6 family member 1 TM4SF1 Membrane Q5U3C3 Transmembrane protein 164TMEM164 Membrane Q8IY95 Transmembrane protein 192 TMEM192 Lysosomemembrane Q8WUH6 Transmembrane protein 263 TMEM263 Membrane Q9BQJ4Transmembrane protein 47 TMEM47 Membrane Q9NW97 Transmembrane protein 51TMEM51 Membrane Q9HCN3 Transmembrane protein 8A TMEM8A Membrane Q6UWD8Transmembrane protein C16orf54 C16orf54 Membrane Q13641 Trophoblastglycoprotein TPBG Cell membrane Q9BTW9 Tubulin-specific chaperone D TBCDCell junction P50591 Tumor necrosis factor ligand TNFSF10 Membranesuperfamily member 10 Q92956 Tumor necrosis factor receptor TNFRSF14Membrane superfamily member 14 P25445 Tumor necrosis factor receptor FASCell membrane superfamily member 6 Q6RW13 Type-1 angiotensin IIreceptor- AGTRAP Endoplasmic reticulum associated protein membraneP23458 Tyrosine-protein kinase JAK1 JAK1 Endomembrane system P43405Tyrosine-protein kinase SYK SYK Cell membrane Q01973 Tyrosine-proteinkinase ROR1 Membrane transmembrane receptor ROR1 P07947 Tyrosine-proteinkinase Yes YES1 Cell membrane P18031 Tyrosine-protein phosphatase non-PTPN1 Endoplasmic reticulum receptor type 1 membrane P78324Tyrosine-protein phosphatase non- SIRPA Membrane receptor type substrate1 O95164 Ubiquitin-like protein 3 UBL3 Cell membrane Q6ZWK4Uncharacterized protein C1orf186 C1orf186 Membrane P46939 Utrophin UTRNCell junction Q9P253 Vacuolar protein sorting-associated VPS18 Lateendosome membrane protein 18 homolog Q9UK41 Vacuolar proteinsorting-associated VPS28 Cell membrane protein 28 homolog Q9H9H4Vacuolar protein sorting-associated VPS37B Late endosome membraneprotein 37B Q9UN37 Vacuolar protein sorting-associated VPS4A Prevacuolarcompartment protein 4A membrane O75351 Vacuolar proteinsorting-associated VPS4B Prevacuolar compartment protein 4B membraneO95562 Vesicle transport protein SFT2B SFT2D2 Membrane O95292Vesicle-associated membrane VAPB Endoplasmic reticulumprotein-associated protein B/C membrane P21281 V-type proton ATPasesubunit B, ATP6V1B2 Endomembrane system brain isoform Q9Y6M5 Zinctransporter 1 SLC30A1 Cell membrane Q9ULF5 Zinc transporter ZIP10SLC39A10 Membrane Q13433 Zinc transporter ZIP6 SLC39A6 Cell membrane

The invention claimed is:
 1. A method for assaying a fluid sample from a patient, the method comprising: assaying the fluid sample for presence of extracellular vesicles comprising MT-C02 (mitochondrially encoded cytochrome c oxidase II) and COX6c (cytochrome c oxidase subunit 6C), and wherein the patient has or is suspected of having a cancer.
 2. The method of claim 1, wherein the fluid sample is blood or a fraction thereof.
 3. The method of claim 1, wherein the fluid sample is serum.
 4. The method of claim 1, wherein the fluid sample is plasma.
 5. The method of claim 1, wherein the fluid sample is ovarian cystic fluid.
 6. The method of claim 1, comprising isolating the extracellular vesicles prior to the assaying.
 7. The method of claim 1, wherein assaying comprises detecting MT-CO2 and COX6c using an epitope specific binder for MT-CO2 and an epitope specific binder for COX6c.
 8. The method of claim 7, wherein the detecting comprises detecting MT-CO2 and COX6c using ELISA.
 9. The method of claim 1, wherein assaying comprises capturing the extracellular vesicles by using the epitope specific binder against MT-CO2 and detecting the extracellular vesicle using the epitope specific binder against COX6c.
 10. The method of claim 9, wherein the detecting comprises detecting MT-CO2 and COX6c using ELISA.
 11. The method of claim 1, wherein assaying comprises capturing the extracellular vesicles by using the epitope specific binder against COX6c and detecting the extracellular vesicle using the epitope specific binder against MT-CO2.
 12. The method of claim 11, wherein the detecting comprises detecting MT-CO2 and COX6c using ELISA.
 13. The method of claim 7, wherein the epitope specific binder is an antibody, an aptamer, and/or a peptide.
 14. The method of claim 13, wherein the epitope specific binder is an antibody.
 15. The method of claim 1, wherein the cancer is melanoma.
 16. The method of claim 1, wherein the cancer is breast cancer.
 17. The method of claim 1, wherein the cancer is ovarian cancer.
 18. An apparatus for implementing the method of claim
 1. 19. A computer program product comprising non-transitory computer-readable storage media having computer-readable instructions stored thereon, the computer-readable instructions being executable by a computerized device comprising processing hardware to execute the method of claim
 1. 